orthobiologics an ossap monograph

ORTHOBIOLOGICSan OSSAP Monograph

Copyright C Orthopaedic Surgeons Society of Andhra Pradesh, 2019Cover Design : Editorial Team

Typesetting & Printed at :

Arudra Printing4/7 Brodipet, Guntur-2.Ph : 9246767666

This edition is for distibution by OSSAP and Janssen, Not for sale.

This book is dedicated to my parents Dr. S. Brahmanandam & Dr. I.A. Kanthamma

and my Teacher Dr. G. Narasimha Reddy

- Dr. Amarnath Surath Chief Editor

I Message from President 5

II Message from Secretary 7

III From the Editorial Team 9

IV Contributors 11

V Foreword 13

1. Introduction to Orthobiologics 15

2. BMPs in Orthopaedics 17

3. Platelet biology and introduction of Platelet Rich Plasma 29

4. Platelet Rich Plasma extraction with low cost assembled kit 37

5. PRP in Tendinopathies 45

6. PRP in OA Knee : Consensus Document 49

7. Bone Marrow Aspirate Concentrate (BMAc) 53

8. BMAc in Non union of Fractures 61

9. Mesenchymal Stem Cells in Management of 69Avascular Necrosis Femur Head

10. Orthobiologics in the treatment of Early Osteoarthritis - 77a Systematic Review

11. Bibliography 89

Contents...

President's Message“it’s what you think we know that keeps us from learning”

- Claude Bernard (19th century French Physiologist)Orthobiologics is a fascinating subject everybody

thinks they already know and I am no exception. Inthe world of orthopaedics, replacing everything is anoverwhelming practice. Regenerating biology is likea miracle. In the past it was too much to ask for andit was almost a weird thing. The dominance of industryand orthometallurgists has subdued any idea otherthan replacement.

But everything will fall or rise to its right place in time. At present, new inroads and new thought process have paved the way for use of biologics inmedicine in general. Orthopaedics is one of the main branches of the medicineutilizing biologics to answer many unsolved questions .

We are bound to fail when you do not know for sure and do not know howto execute. This is where this monograph “ORTHOBIOLOGICS” will help indecision making and management. When we proposed the idea of bringing amonograph from OSSAP all fingers pointed to Professor Dr. Amarnath, thescientific orthopaedic surgeon from Guntur. Professor Amarnath and his teamDr.Dakshina Murthy, Dr.Ramireddy and Dr. Naresh Babu have unveiled a miracle(I feel Biologics is a miracle) for OSSAP .

As President of OSSAP, I am very fortunate to be associated with the teamin bringing out the first monograph from OSSAP. All the members of OSSAPgenuinely applaud the whole hearted efforts put in by the editorial team andauthors of various chapters in the monograph.

I wish that the future administrative body of the OSSAP will continue thisAcademic feature.

Dr. Y. Nageswara RaoPresident, OSSAP

It gives me immense pleasure to present beforeyou “The OSSAP Monograph” series. The monographon Orthobiologics”, is first of its kind from our statechapter. I take this opportunity to thank our Hon’blePresident, Prof. Y Nageswara Rao for the initiation ofOSSAP Monographs. I congratulate Prof. S.Amarnath (Editor in Chief), Sub-Editors and allauthors of individual chapters for striving hard inbringing this extremely rare topic into existence.

Orthobiologics is in itself a complex topic in the post-graduation phase and withthe advent of this book, post-graduates can get their basics right.

This Monograph comprises of interesting chapters like Bone MorphogenicProteins and Platelet Rich Plasma, which are produced in a very logistical andeconomical manner. I would like to recommend this book for all the orthopaedicsurgeons for updating their knowledge on Orthobiologics.

GrGrGrGrGreetings freetings freetings freetings freetings from the ofom the ofom the ofom the ofom the offffffice ofice ofice ofice ofice of secr secr secr secr secretaretaretaretaretary ofy ofy ofy ofy of OSSAP OSSAP OSSAP OSSAP OSSAP

Dr. Naresh Babu JSecretary, OSSAP

We wish to thank OSSAP for giving us the opportunity to compile thefirst monograph brought out by the association. Much thought has gone intoselecting Orthobiologics as the topic since there are many lacunae in ourknowledge regarding these substances. The line between proven methodologiesand experimental work is blurred, hence we wish to set the record straight withthis endeavour. Each chapter has been selected with care so that most of theclinically relevant aspects are covered.

The contributors were chosen based on their work in their respective fieldsand have divulged nuggets of information which will be helpful to all. This bookwill be of equal help to the practicing surgeon and post graduate student alike.Wherever needed technical details have been provided to initiate surgeons intothe fascinating world of regenerative medicine. The team will be most gratifiedif some of the younger surgeons are stimulated to take up full time research intoorthobiologics.

We wish that this monograph is followed by many more and hope the readerswill find the book academically useful and reliable in clinical practice. Our heartfeltthanks to OSSAP president Dr. Y. Nageswara Rao and Secretary Dr. NareshBabu for giving us this opportunity and Janssen for sponsoring the monograph.

From the Editorial Team…

Dr Amarnath SurathDr AV Dakshina Murthy Dr Mettu Rami Reddy

Dr J Naresh Babu

Dr. Rami Reddy Mettu Dr. AV Dakshina Murthy Dr. J Naresh BabuDr. Amarnath Surath

Contributors...

Dr. Amarnath SurathProf. & HOD Department of Orthopaedics, NRI Medical College, Guntur.Amar Orthopaedic Hospital, Gunture-mail : [email protected]; Mob : 9399962062

Dr. Rami Reddy MettuAssociate Prof. Department of Orthopaedics, NRI Medical College, Guntur.MGR Multispeciality Hospital, Gunture-mail : [email protected]; Mob : 9985314900

Dr. AV Dakshina MurthyOrthopaedic Consultant, Gunture-mail : [email protected]; Mob : 9849127560

Dr. Arun Kumar ViswanadhaConsultant Spine Surgeon, Mallika Spine Centre, Kothapet, Guntur.e-mail : [email protected]; Mob : 7660076007

Dr. J Naresh BabuConsultant Spine Surgeon, Mallika Spine Centre, Kothapet, Guntur.Secretary OSSAPe-mail : [email protected]; Mob : 9989426898

Dr Vijay Shetty

Dr. Karthik PingleConsultant Orthopaedic Surgeon, Apollo Health City, Hyderabad.E-mail : [email protected]; Mob : 9959020117

Dr. Ramesh SenSenior Director and Head of Department- Institute of Orthopedic SurgeryMax Hospital, Mohali, Haryana.Ex-Prof. PGIMER Chandigarh.e-mail : [email protected]; Mob : 9815856677

Dr. K Satya KumarProf. Department of Orthopaedics, NRI Medical College, Guntur.Orthocare, Vijayawadae-mail : [email protected]; Mob : 9000366555

Contributors...

Consultant Orthopaedic Surgeon, Hiranandani Hospital, Mumbai. IndiaVice President, Indian Association of Sports MedicineSecretary, Indian Biologics Orthopaedics Society (IBOS)Associate Editor, SICOT World (open access) Journal, Examiner, SICOT Internationalexaminations, Member, Editorial Board, Hip International,Member, Editorial Board, Asian Journal of Arthroscopye-mail : [email protected]; Mob : 9920707771

Foreword

Thank you very much for asking me to write the foreword. The scienceof orthopaedics has had many twists and turns in my career spaning fivedecades. From initial conservative management to aggressive internalfixations, and now harnessing the body's internal mechanisms to repair itselffrom within is facinating. This is the magical world of Orthobiologics.

The concept of this monograph is very thoughtful of OSSAP as thisbook provides a valuable single reference on this relatively new topic. I laudthe efforts of the Executive body and the Editorial Team in bringing out thisexcellent book.

Dr. C.K. Sarma, M.S.

Rtd. Professor of Orthopaedics

Introduction to Orthobiologics

Orthobiologics are a group ofsubstances found in the body whichcan help the process of healing orregeneration. They may be genes,proteins, cells or fully formed tissues.The beneficial effect may manifest inconcentrations many times higher thanfound physiologically in the body. As agroup, orthobiologics include BoneMorphogenetic Proteins ( BMP ),Growth Factors, Stem Cell Therapy,Synthetic Bone Graft and AllograftTissue.

Every couple of decades brings tolight a new paradigm in orthopaedicmanagement. The seventiesrevolutionized fracture fixation by AO/ASIF group, eighties saw phenomenalgrowth in arthroplasty and themillennium belongs to RegenerativeMedicine of which Orthobiologics, forman integral part. The trend is toconserve biology and enhance tissuerepair or regeneration by stimulatingnatural signaling mechanisms. Themethodologies described herein areeither specially developed molecules orcells whose numbers are increasedmany fold to have beneficial effects.

Bone Morphogenetic Proteins(BMP), discovered by Marshal Urist in1965 were received with greatenthusiasm due to their potential forosteoinductive potential. Proteinsapproved for clinical use are BMP-2(Infuse ) and BMP-7 (OP-1). They arecommercially synthesized byrecombinant technology and are veryexpensive, which is why they are notin widespread use. Classic indicationsfor BMP use are in management ofopen fractures, nonunion and spinalfusion.

Bone Marrow AspirateConcentrate (BMAc) has gainedpopularity over the years due to thehigh concentration of stem cells andthe regulatory issues surrounding theuse of cultured stem cells. BMAC isautologous, implanted into site ofpathology without any modification.The procedure is performed on site,which reduces potential complicationssuch as contamination and infection.Common applications of BMAC are inthe treatment of non union of fracturesand avascular necrosis of femoralhead.

Dr. Amarnath Surath

16 Orthobiologics

Platelet Rich Plasma ( PRP ),has created a hype unparalled inorthopaedic surgery. The reason forthis meteoric rise in popularity isbecause it was endorsed by highprofile sports persons as a “magicbullet” for musculoskeletal pathology.The other advantage of PRP is easeof administration as an out patientprocedure. Many specialities havefound a myriad of uses for PRP, suchas dentistry, cosmetology, dermatologyand ophthalmology. PRP as a singleentity has managed to highlight the

importance of orthobiologics as awhole.

3D Tissue printing is the ultimatefrontier of regenerative medicine.Imagine being able to design a wholeorgan or parts required, which candirectly substitute damaged ordiseased parts without risk of rejectionor foreign body reaction. Achieving thisfeat will be an ode to the ingenuity ofmankind and that can be madepossible only by the use ofOrthobiologics.

17

BMPs in OrthopaedicsDr. Naresh Babu J. & Dr. Arun Kumar Viswanadha

Bone Morphogenic Proteins(BMPs) are a group of molecules whichinduce mesenchymal stem cells todifferentiate into bone forming cell linesthat form new bone. They are a groupof noncollagenous glycoproteins thatmostly belong to the transforminggrowth factor-beta (TGF-b)superfamily. BMPs have been widelyused in the field of orthopaedics sincethe last two decades. Marshall Urist in1965 initially described theosteoinductive activity of BMPs. BMPsare a group of osteoinductive proteinsthat are usually extracted from bonematrix which are responsible forskeletal regeneration and bonehealing. BMPs are capable ofinhibiting chondrocyte differentiationindependently and are recognised fortheir regulatory effects, especially inembryonic phase in the form of growth,differentiation and morphogenesis. Theprimary functions of BMPs are toactivate inactive mesenchymal stemcells and to differentiate them into

osteoblast, chondroblasts andfibroblasts.

Apart from the above functions,BMPs are also involved in otherphysiological and pathologicalprocesses such as inflammatoryresponse, bone formation andresorption, growth signalling pathways,oncogenesis and immune response.The safety and efficacy of BMP asbone graft substitute are influenced byits purity, local effects, systemic effects,immunogenicity and biocompatibility.

Introduction

Bone ossification occurs throughboth intramembranous ossification andendochondral ossification.

Intramembranous ossification –Primitive mesenchymal stem cells(MSCs) are transformed intoosteoprogenitor cells and then intoosteoblasts which ultimately form theosteoid. It is typically seen in skull,mandible and clavicle bones.

18 Orthobiologics

Endochondral ossification – Herethe mesenchymal stem cells transforminto chondroblasts which lays downcartilage which later matures anddegenerates. This degeneratedcartilage is invaded by blood vesselsand osteoblasts which finally lay downosteoid.

Both cellular events ofintramembranous ossification andendochondral ossification involveMSCs which may be bone marrowderived or periosteum derived. Theseprimitive MSCs are pluripotentprogenitors that can differentiate intoboth osteoblasts and chondroblasts.This differentiation is regulated bymolecules such as BMPs andFibroblast growth factor (FGF).

Bone formation duringdevelopmental phase and fracturehealing phase is governed by similarcellular and molecular events. Fracturehealing requires adequate growthfactors, sufficient bone matrix, goodmechanical stability which altogetherconstitute appropriate cellularenvironment. When the process offracture healing fails leading to non-union or delayed union, it requires

some stimulation for the process ofbone formation. This can be achievedby biophysical methods such asultrasound or biological interventionssuch as bone graft, bone marrow orbiologically active molecules.Autogenous bone grafts are capableof stimulating bone formation throughosteogenesis, osteoinduction andosteoconduction. Osteogenesis is theprocess of direct bone formation by theliving osteoblasts present in the graft.Osteoinduction is defined as the abilityof factors to induce osteogenesis in theextra osseous site. Osteoconduction isthe process of allowing bone formationon its surface in order to promote bonegrowth. Autogenous bone graft fromiliac crest is considered to be goldstandard in the treatment of non-unions, delayed unions and bonedefects. However, morbidity of graftsite, limited availability of bone graftand variable success rates of unionmandates the need for better options.

One of the effective method ofavoiding these complications arisingfrom autogenous bone graft harvest isby the use of bone graft substitutes inthe form of BMPs due to theirosteoinductive properties.

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Structure

TGF-b superfamily constitutes ofvarious growth factors anddifferentiation factors, with BMPsforming the largest part in the familythat also comprises of activins andinhibins. Currently there are more than20 known BMPs and two commerciallyavailable BMPs - recombinant BMP-2(rhBMP-2) and recombinant BMP-7(rhBMP-7) approved by US FDA in2004. In human bones, BMPs aresecreted by osteoprogenitor cells,osteoblasts and platelets. BMPs aresynthesized as inactive precursorproteins which contain propeptides andhydrophobic leader sequence. Theactive portion of BMPs is howeverlocated at the carboxy terminal ofprecursor molecule. This biologicallyactive portion is released by proteolyticremoval of signal peptide and pro-petide. Active portions of all BMPscontain seven cysteine amino acidresidues which are positioned similarto other members of TGF- superfamily.Of the seven cysteines, six formintramolecular disulphide bondswhereas seventh cysteine residue isinvolved in dimerization with anotherBMP monomer through a covalent

disulphide bond, resulting in abiologically active dimeric ligand forreceptor activation. Overall the corestructure of BMP dimers consists of“cysteine-knot” structure and istypically described as “wrist andknuckle” or “two bananas” shape.

BMPs, like any other TGF-b familymembers elicit their effects through twotypes of serine-threonine kinasetransmembrane receptors, type I andtype II receptors. But unlike TGF-b,BMPs are capable of binding to type Ireceptors even in the absence of typeII receptors. However, their bindingaffinities increase when both type I andtype II receptors are present

Classification of BMPs

Till date more than 20 BMPs havebeen identified in vertebrates. Basedon structural homology, BMPs can befurther classified into severalsubgroups which includes BMP-2/-4group, BMP-5/-6/-7/-8 group, BMP-9/-10 group and BMP-12/-13/-14 group.Among all BMPs, only BMP-1 has ametalloprotienase structure and actsas a carboxy terminal propeptidase fortype I collagen. BMP family membersare also identified in invertebrates such

BMP's in Orthopaedics

20 Orthobiologics

as Drosophila decapentaplegic, 60A/glass bottom boat which are structurallysimilar to BMP-2/-4 and BMP -6/-7 respectively.

Functions of BMPs

The actions and functions of BMPsdepend on various factors which are:

1. Type of target cell.

2. Maturation phase of target cells.

3. Local concentration of BMPs.

4. Other biological signals.

BMPs are mitogens (growthfactors) that stimulate the productionand multiplication of morphogens(differentiation factors) that transformconnective tissue cells intoosteoprogenitor cells. They play apivotal role in embryonic developmentthrough specification of positionalinformation in the embryo. Theyregulate growth, differentiation,

Figure 1 – Interrelations of BMP family. One subgroup consists of BMP-2,BMP-4 and Drosophilia decapentaplegic(dpp). Other subgroup consists of

human BMP-5, BMP-6, BMP-7, BMP-8 and Drosophilia 60A.

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chemotaxis and apoptosis of cells suchas epithelial, mesenchymal,haematopoietic and neuronal cells.Apart from inducing bone formationthrough intramembranous orendochondral ossification, they alsoinduce osteoclasts leading to boneresorption. However, the local effects

of BMP are regulated by number ofextracellular and intracellularantagonists (Table 1). Extracellularantagonists form complexes with BMPand prevent them from binding to theirreceptors whereas intracellularantagonists interfere with activation ofR-Smads or facilitate their degradation.

Carriers of BMPs

BMP is a water soluble moleculewhich diffuses into the body fluid easily.When administered alone, most of theprotein molecule is lost rapidly due todiffusion and irrigation. To negate theseeffects and have a prolonged localisedeffect at target site, BMPs areadministered in a carrier. Type Icollagen is the most preferred carrierused for transport of BMPs. Type I

Table 1 – Antagonists of BMPs

collagen can be extracted from boneor tendons, BMPs bind tightly tocollagen extracted from bone and lesstightly to collagen extracted fromtendons. Also compression of collagencarrier leads to rapid release of BMPs,thereby the carrier is usually protectedby a cage. Other carriers or deliverysystems used previously in theliterature are summarised in table 2.

Extracellular antagonists Intracellular antagonists

noggin Smad6

chordin Smad7

twisted gastrulation (Tsg) Smad8b

gremlin Smurf1

follistatin Smurf2

BMPER


22 Orthobiologics

Metallic implants Ceramics NaturalPolymers

Syntheticpolymers

Others

Anodic oxidized

nanotubular Ti

implant

Surface

mineralized

Ti6Al4V

rhBMP-2 and

heparin

immobilized Ti

BMP-2

incorporated on

HA coated

Ti surface

HA Granules;

300-500 lm

β−TCP

Granular implant

(porosity: 75%,

pore size: 50-

350 mm)

Fibrous glass

membrane

Mesoporous

silica bio-glass

scaffold

Collagen

Gelatin

Chitosan

Keratinbased

Hyaluronicacid

Silk based

Polyethyleneglycol

Polylactic acid

Poly (lactic-co-glycolic acid)

Poly e-caprolactone

Compositematerials

Stimulusresponsivepolymer

Stimulusresponsivepolymer

pHresponsivepolymer

Clinical Applications inOrthopaedics

BMPs are usually applied directlyas an adjuvant therapy or as analternative to autograft. However dueto high costs being involved, its clinicalusage has been limited. Currently, twocommercial forms of BMPs areavailable for clinical usage – rhBMP-2and rhBMP-7. While rhBMP-2 is FDAapproved in 2002, while rhBMP-7

carries only humanitarian deviceexemption status.

1. BMP as an alternative to autograftfor non-unions

Long Bones – Currently, US Foodand Drug Administration (FDA)approval of BMP use in long bones islimited to tibia non-unions asalternative to autograft. Many authorsbelieve that despite the technicaldifference at different sites, non-union

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is similar between upper and lowerlimbs. However, literature suggeststhat results with usage of BMPs inupper limbs has been poor. Apart fromits usage in humerus pseudoarthrosis,BMPs have also been successfullyused in few clinical situations involvingclavicle and ulna.

Pseudoarthrosis of tibia remainsto be the most frequent site of longbone non-union due to its poormuscular coverage, high incidence ofopen injuries and its anatomicalpeculiarity. Friedlaender et al initiallyreported the efficacy of BMP-7 in thetreatment of tibia non-unions. Total of124 tibia non-unions were treated withrecombinant human osteogenicprotein-1 (rhOP-1 or BMP-7) in a typeI collagen carrier or by fresh boneautograft. According to them, 81% ofOP-1/BMP-7 treated tibia non-unionsand 85% of those receivingautogenous bone were successfullyhealed. They finally concluded thatBMP-7 provided comparable resultswhen compared with bone autograft,without donor site morbidity. This paperhas reinforced interest in others and ithas been accepted to be an alternativeto autograft in long bone non-unions.

However, it is absolutely necessary torespect the classic principles ofsurgical treatment for pseudoarthrosis.

Scaphoid – Scaphoid is verycommonly affected with non-union dueto its precarious vascular supply. Bilicet al in a randomised control trialcompared the efficacy of BMP-7 in themanagement of scaphoid non-unions.One group was treated by autograftalone, second by BMP-7, third byautograft with BMP-7 and fourth byallograft with BMP. Treatment includedfixation with Herbert Screw and plastercast for 1 month. Patients treated withautograft and BMP-7 healedsuccessfully within 3 months whereasother groups required 9 months.However, all the evidences onscaphoid non-unions were poor.

2. BMP as an alternative to autograftfor fusion

Foot and ankle – Few authorshave used BMPs to improve the ratesof union in ankle arthrodesis forpatients with comorbidities affectingbony union. Most publications deal withdifferent types of arthrodesisperformed in complex cases. Initiallyuse of BMP in complex foot and ankle


24 Orthobiologics

arthrodesis seem to be a safe optionconsidering its high failure rates.However, there is no randomisedcontrol trial available till date to give asuggestable conclusion.

Spine

By far, spine is the most commonsite associated with BMP application.US FDA has approved the usage ofrhBMP-2 in anterior lumbar interbodyfusions. However recent studies haveshown that BMP use has beenextended to fusions performed withother approaches as well.

Studies leading to US FDAapproval of rhBMP-2 in SpineSurgery

After conducting various animaltrials, Boden et al in the year 2000 firstpublished a randomised control trialwith usage of rhBMP- 2/collagensponge as a substitute for autologousbone graft for anterior lumbar interbodyfusion (ALIF). They reported that spinalfusion was equally reliable with usageof rhBMP-2 when compared with iliaccrest autograft without any adverseeffects. In 2002, Boden et al reporteda second study on rhBMP-2 wherethey demonstrated consistent

radiographic fusion rates whounderwent posterolateral fusion with orwithout internal fixation. In the timespan of 10 years, a total of 13 industrialsponsored trials were published whichevaluated the efficacy and safety ofrhBMP-2 in lumbar and cervical spinesurgery. Total of 1580 patients wereenrolled in all studies (780 – rhBMP-2group and 800 – control group). In theinitial trials, rhBMP-2 was administeredin two different preparations;

InFUSE (Medtronic SofamorDanek, Memphis, TN) – 1.5mg/mL ofrhBMP-2

AMPLIFY (Medtronic SofamorDanek, Memphis, TN) – 2mg/mL ofrhBMP-2

All these human studiesrecommended the clinical applicationof BMPs in spine surgery. Theyreported more than 95% fusion ratesat last follow up across all fusiontechniques. They further stated thatrhBMP-2 was superior or equallyeffective to iliac crest autograft in termsof clinical outcomes without anyadverse effects.

Based on these clinical studies,

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US FDA finally approved InFUSE(Medtronic Sofamor Danek, Memphis,TN) in the year 2002 as a bone graftsubstitute for a single level ALIFbetween L4-S1 within a specific LT-cage (LT-CAGE; Medtronic SofamorDanek, Memphis, TN). Initiallydescribed as an adjuvant for spinalarthrodesis more widespread usage ofInFUSE has been documented in thelast decade. In USA, usage of InFUSEhas increased from 0.7% in 2002 to25% in 2006. By the end of 2007, 50%of all primary anterior lumbar interbodyfusions, 43% of posterior/transforaminal lumbar interbodyfusions, 30% of posterolateral fusionswere reported with InFUSE. Howevermore than 85% of its utilisation wasaccounted for off-label administration.

Controversy with BMPs inSpine Surgery:

Not a single adverse effect wasreported in the 13 industry sponsoredtrials which were published in manyreputed journals. The estimated risk ofrhBMP-2 usage has been less than0.5% which is far inferior to commonlyused analgesics and antibiotics. Thesetrials also reported high rates ofmorbidity (40%-60%) with iliac crest

bone graft harvest. However, severalindependent studies started reportingserious complications with rhBMP-2usage from the year 2006 with adverseeffects ranging from 20% to 70%.These complications includeheterotropic ossification, increasedinfection rates, formation of seroma/haematoma, dysphagia, difficulties inpost-operative airway maintenance,increased incidence of neurologicaldeficits, increased post-operativepains, retrograde ejaculation andmalignancy (Table 3). In particular,there has been increased associationbetween retropharyngeal edema andrhBMP-2 which led to difficulties withairway maintenance. Such reportshave mandated US FDA to issue apublic health notification concludingthat safety and efficacy of rhBMP-2 incervical spine was not established.

Following this public healthnotification, Federal Governmentlaunched an investigation into thereported off-label usage of InFUSE andclaims of illegal marketing by Medtronicwhich includes “payments to doctorsin form of cash/gifts to use of InFUSE”.In 2011, Carragge et al compared theefficacy reports of rhBMP-2documented in original industry trials


26 Orthobiologics

with FDA data summaries, follow upstudies and administrative databases.They concluded that adverse reactionsin patients receiving rhBMP-2 was 10to 50 folds higher than originallyreported. They also commented thatsignificant financial relationship existsbetween authors of 13 original FDAtrails and Medtronic company (Total

sum involved - $12,000,000 to$16,000,000; Range - $560,000–$23,500,000 per study). For studiesreporting on 20 or more patients withusage of rhBMP-2, at least one authorreceived $1,000,000. Whereas if thestudy includes more than 100 patients,amount increased to $10,000,000.

Table 3 - List of potential adverse events associated with the use ofINFUSE Bone Graft/LT-Cage Device

Bone fractureBowel or bladder problemsCessation of any potential growth of the operated portion of the spineChange in mental statusDamage to blood vessels and cardiovascular system compromiseDamage to internal organs and connective tissueDeathDevelopment of respiratory problemsDisassembly, bending, breakage, loosening, and/or migration of componentsDural tearsEctopic and/or exuberant bone growthFetal development complicationsForeign body (allergic) reactionGastrointestinal complicationsIncisional complicationsInfectionInsufflation complicationsLoss of spinal mobility or functionNeurological system compromiseNonunion (Pseudarthrosis), delayed union, mal-unionPostoperative change in spine curvature, loss of correction, height, and/or reductionRetrograde ejaculationScar formationTissue or nerve damage

27

3. BMPs in experimental phase

Besides all the above clinicalapplications, experimental studies areundergoing with usage BMPs incartilage repair. Many authors believethat use of BMPs in paediatric

population is contraindicated.However, few studies are inexperimental phase with use of BMPsin conditions like congenitalpseudoarthrosis of tibia and LeggCalve Perthes disease.

Conclusion

The accumulation of data on BMP’s provides a number of key lessonsto orthopaedic surgeons. Firstly, use of BMP’s has been associated withhigh fusion rates, both in non-union of tibia and in spinal arthrodesis.However, with bone graft harvested from iliac crest still being consideredas gold standard for achieving union in long bones and with local graftobtained being sufficient for arthrodesis in spine surgery, usage of BMP’sis being limited in today’s practice. Secondly, surgeons should be awareof devastating complications which can be encountered with irresponsibleusage of BMP’s. Finally, the orthopaedic community and patients mustremain informed about the profile of these BMP’s which can offer benefitswhen used cautiously and responsibly.


28 Orthobiologics

29

Platelet biology and introduction to Platelet Rich Plasma

a nucleate, they have distinctmitochondria, plasma membranecomposed of phospholipid bilayer.Thisisthe site of expression of varioussurface receptors and lipid rafts whichhelps in signalling and intracellulartrafficking.

Platelets contain huge number ofbiologically active molecules withincytoplasmic granules namely alphagranules(α), dense granules(δ) andlysosomes(λ). The contents of thesegranules are released when plateletsare activated during vessel injury andthus play an important role inhaemostasis, inflammation, woundrepair and also in pathological processof atherosclerosis. Each formedplatelet contains about 50-80 α-granules. These α-granules containmore than 30 adhesive proteinsincluding platelet derived growth factor(PDGF), transforming growth factor(TGFβ, β1 and β2 isomers), plateletfactor 4 (PF4), interleukin1 (IL1),platelet-derived angiogenesis factor(PDAF), vascular endothelial growthfactor (VEGF), epidermal growth factor(EGF), plateletderived endothelialgrowth factor (PDEGF), epithelial cellgrowth factor (ECGF), insulin likegrowth factor (IGF) etc.

Dr. Rami Reddy Mettu

Biology of Platelets

Platelets were initially discoveredby GiulioBizzozero in the year 1882.For many decades, the dynamic andmultifunctional nature of plateletsremained a field of interest mostly forbiologists and not clinicians. Althoughvery dynamic in functional activity, theyusually prefer to remain in inactivestate and get activated only when ablood vessel is damaged or injured.Haemostasis is not the sole functionof platelets, they possess severalmultifunctional attributes monitoringthe homeostasis of the body.

Platelets develop frommegakaryocytes in the marrow. Eachmegakaryocyte can produce 5000-10000 platelets. Approximately 2/3rd ofthe platelets circulate in the blood and1/3rd are stored in the spleen. Anaverage healthy adult can produce 1011

platelets per day. The normal plateletcount is (150– 400) × 103 per microliterof blood. The diameter of a matureplatelet is 2-3 5μ. Platelets usuallyremain alive for 5-11days.Old plateletsare destroyed by phagocytosis in thespleen and liver by Kupffer cells.

Platelets are unique in theirstructural assembly. Though they are

30 Orthobiologics

Platelet Rich Plasma

By definition, Platelet Rich Plasma (PRP) must contain a higherconcentration of platelets than baseline. To be labeled as PRP, a platelet countof 3-5 times of the baseline should be present in the platelet concentrate(manystudies like Max et al mention the platelet count 1 million as standard valve forPRP).

The properties of PRP are based on the production and release of thefactors when the platelets are activated. Platelets begin secreting theseproteins within 10 minutes of activation. After initial release of growthfactors, the platelets synthesize and secrete additional factors for theremaining days of their life span.

The rationale for use of PRP is based on their capacity to supply and releasesupraphysiologic amounts of essential growth factors and cytokines from theiralpha granules to provide a regenerative stimulus that augments healing andpromotes repair in tissues with low healing potential (Table 1).

Figure 1: Biology factors in Platelet Rich Plasma.( courtesy of mikel sanchez et al.)

31

Table 1: Growth Factors in Platelet Alpha granules and its function

PDGF Stimulates cell proliferationChemotaxisStimulates angiogenesis

TGF-β Stimulates production of collagen type I and type IIIAngiogenesisRe-epithelializationPrevents collagen break down

VEGF Stimulates angiogenesis

EGF Influences cell proliferation and cytoprotectionAccelerates re-epithelializationIncreases tensile strength in woundsFacilitates organization of granulation tissue

b-FGF Stimulates angiogenesisPromotes stem cell differentiation and cell proliferationPromotes collagen production and tissue repair

IGF-1 Regulates cell proliferation and differentiationInfluences matrix secretion from osteoblastsProduction of proteoglycan, collagen, and othernoncollagen proteins

GROWTHFACTORS FUNCTIONS

Components of Platelet Rich Plasma

1. Platelets

Although platelets play a key rolein haemostasis, they are central inmediating the anabolic effects of PRP

by virtue of releasing growth factorsstored in their alpha granules. Duringthe initial phases of wound repair,activated platelets attract and fostercell migration into the wound byaggregating and forming a fibrin matrix.This matrix then serves as a tissue


32 Orthobiologics

scaffold for sustained release ofplatelet growth factors and cytokines,which stimulate cell recruitment,differentiation, and communication.

2. Leukocytes

Leukocytes are essential mediators ofthe inflammatory response, hostdefence against infectious agents, andwound healing. Neutrophils areinvolved in the inflammation phase ofwound healing. Monocytes andmacrophages facilitate tissue repair bydebriding and phagocytising damagedtissue and debris. Similar to platelets,macrophages also secrete growthfactors that are important in tissuerepair and have been shown tocontribute to subchondral boneregeneration.

3. Red blood cells

Red blood cells (RBC) content is

typically reduced or absent in PRPbecause of the centrifugation process.Destructive process is thought to occurin human synoviocytes treated withRBC concentrates, leading tosignificantly greater cell death andcartilage degradation.

Classification of PRP

Dohan et al in the year 2009classified PRP based on presence ofleukocytes and density of fibrin network(Table 2).Mishra et al in the year 2012proposed a classification system whichis based on platelet count, activationand leukocytes for clinical applicationin Sports medicine (Table 3). Anotherclassification system similar to Mishraet al classification is called “PAWClassification”(Platelets, Activation,White cells). However, Dohan’sclassification is still being widely usedtill date.

P-PRP

Table2: Dohan’s classification of PRP

Pure Platelet-Rich PlasmaOr Leukocyte - PoorPlatelet - Rich Plasma

without leukocytes and with a low-densityfibrin network after activation

L-PRP Leukocyte-andPlatelet-Rich Plasma

leukocytes and with a low-density fibrinnetwork

P-PRF Pure Platelet-RichFibrin

without leukocytes and with a high-densityfibrin network

L-PRF Leukocyte- and Platelet-Rich Fibrin

leukocytes and with a high-density fibrinnetwork

33

Table 3: Mishra’s Classification of PRP for application in Sports medicine.

Types White blood cells Activation of PRP

Type 1 (A/B) L-PRP solution increased No activation

Type 2 (A/B) L-PRP gel increased activated

Type 3(A/B) P-PRP solution Minimal or absent No activation

Type 4(A/B) P-PRP gel Minimal or absent activated

L-PRP: Leukocyte rich platelet rich plasma, P-PRP- leukocyte poor plasma

NOTE: Subtype A – Platelet concentrations >5´ patients baseline. Subtype B – Plateletconcentrations <5 ´ patients baseline.

Sports medicine platelet richclassification system sub divided PRPtype into A&B depending on whetherthe platelet concentration is more than5X patients baseline (A) or less than5X patients baseline (B). This subclassification is weakened by the factthat the serum can influence the finalplatelet count.

PRP Applications

1. Wound Healing

In general, wound healing can beseparated into 3 phases namelyInflammation, Proliferation andRemodeling.The initial inflammationphase is characterized byhaemostasis, with plateletsestablishing clot formation, and the

release of growth factors that aid inactivating and attracting inflammatorycells like neutrophils and macrophagesto the site of injury. The proliferationphase is characterized by theconstruction of an extracellular matrixassociated with granulation,contraction, and epithelialization.Remodelling phase is associated withproduction of collagen and scar tissue.

The physiologic progressionthrough these phases of woundhealing is orchestrated by growthfactors and cytokines, many of whichare released and modulated bycomponents in PRP.

2. Rotator cuff tears

Degenerative changes are


34 Orthobiologics

expected to be the cause of cuff tear,in the older population, There arechances that a meticulously performedrotator cuff repair may fail or heal suboptimally. It is important to note thatthe distal part of the rotator tendon hasinherently poor healing capabilities.Here PRP with its growth factors maythus be an attractive option for thestimulation of tendon healing.

Randelli et al. were the first toconduct an uncontrolled pilot study ofPRP augmentation along witharthroscopic rotator cuff repair. Theirreports showed statistically significantimprovements in VAS, constant andUCLA shoulder scores compared topreoperative values.

Rha et al.compared ultrasoundguided PRP injection with dry needlingand concluded that autologous PRPinjections lead to a progressivereduction in the pain and disabilitywhen compared to dry needling.

However, results of PRP use invarious studies are quite different.Thus evidences available so far do notprovide a clear picture regarding theuse of PRP in rotator cuff tears.

However, PRP may be used inrotator cuff injuries either as an adjunct

to surgery, or as stand alone injections.But prior explanation to the patientabout the prognosis and the limitedavailable evidences will be healthy.

3. Tendoachilles tears

Tendinopathies and tears ofachilles tendon are notorious for nonhealing. PRP use has been attemptedfor enhancing healing at this site.

DeJonge et al. in a study ofchronic tendinopathy (7 cm proximalto the Achilles tendon insertion)injected PRP or saline at that site . Afterthe procedure they subjected thepatients to an eccentric trainingprogram.

One year of follow up showed noclinical and ultra sonographicsuperiority of PRP injection over theplacebo injection.

Owens et al. reported modestimprovement in functional outcome inpatients who had received PRPinjection for mid substance Achillestendinopathy Monto et al. reportedclinical success in resistant Achillestendinosis. The improvement notedwas in the AOFAS score.

Enhancement of repair of theAchilles tendon tear with PRP has also

35

been attempted. Sanchez et alshowed better results with TA repairwith augmented with PRP in sportspeople In summary, the results of useof PRP in Achilles Tendonopathy aresuperior to their use in complete tearof the tendon.4. ACL reconstruction5. Subacromial impingement6. Shoulder osteoarthritis7. Osteoarthritis knee

Growth factors in the alphagranules have been postulated to bechondroprotective and capable ofimproving the physiology inosteoarthritic joints. Spaková et al.conducted a RCT on 120 patients withKellgren and Lawrence Grades 1, 2,or 3 Osteoarthritis, comparing PRPinjection with hyaluronic acid andconcluded that autologous PRP wasan effective and safe method in thetreatment of the initial stages of kneeosteoarthritis. Patel et al. published theresults of a randomised control trialconducted on 78 patients (156 knees)with bilateral osteoarthritis andreported improvement VAS scores forpain and in all parameters of WOMACscores in patients who received PRPinjections.

Osteoarthritis of the knee is oneof the commonest degenerativediseases encountered in clinicalpractice and the prospect of arrestingthe disease process in its initial stageslooks rewarding. The clinical evidencesof PRP use in early knee osteoarthritisweighs in favour of PRP.

8. Plantar fasciitis

Shetty et al found no difference ineffectiveness of platelet-rich plasmaand corticosteroid injections at 6months of follow-up.

Wilson et al in his one year followup study of showed PRP is moreeffective than cortico-steriods .

Based on above studies it isobvious using PRP in Chronic PlantarFasciitis requires more evaluation

However it can be tried as analternative to coticosteriods.

9. Lateral epicondylitis

This condition can potentially behandled well with conventionalmethods. However a significantnumber of patients do becomeresistant; PRP has significant benefitin this patients.

Mishra and Pavelko were the first


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to use PRP injection for such resistantlateral epicondylitis. Their pilot studyshowed good results in terms of painrelief and functional improvementPeerbooms et al.in his RCT with 100patients, reported better improvementover the period of one year in themanagement of Lateral epicondylitiswith PRP than management withsteroids Chaudhary et al. usedultrasonographic guidance for injectionand noticed a trend towards anincrease in the vascularity at themusculotendinous junction of theextensor tendons.

With available evidences PRPinjection is the treatment of choice,instead of a corticosteroid injection inpatients with failed conventionalmethods.

10. Patellar tendinosis

Jumper’s knee in athletes is acommon cause of knee pain Filardo et

al. evaluated the efficacy of PRPinjections for refractory patellartendinopathy and concluded that PRPinjections have the potential to promotethe achievement of a satisfactoryclinical outcome, even in chronicrefractory tendinopathy.11. Nonunion of bones

Fracture healing is a processaffected by many factors. AlthoughPRP has been reported in literature tobe a biological treatment whichincreases healing, Sayetal reportedadequate healing was not achieved inthe treatment of non-union with PRPinjection. However, in selected patientswith delayed union, PRP injection canbe recommended in non-surgicaltreatment. Sebastian Lippross in hishypothesis opines that PRP can be asupportive procedure in non-union, ifused in the right manner.

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Introduction

Platelet-rich plasma (PRP) isdefined as the plasma fraction derivedfrom autologous blood having a plateletconcentration approximately five timesabove baseline. It is classified as an“Orthobiologic”; a substance thatenhances the body’s innate ability torepair and regenerate. PRP therapyhas lately gained attention as a safe,nonsurgical, biological treatment ofosteoarthritis and a wide variety ofmusculo skeletal conditions. Atpresent there is lack of data availableto confirm that PRP works aspostulated. Future large randomizedcontrolled trials (RCTs) are needed toassess its efficacy. The present studyhas used the gravitational plateletsequestration ( GPS ) technique toconcentrate platelets. We havedesigned a low cost kit by modifyingreadily available consumables and atable top laboratory centrifuge. Afterreviewing the existing literature theparameters for centrifugation weredetermined to produce a platelet countclose to one million. These kits providePRP at economic price so as to make

large scale clinical trials possible.

Materials and methods

A total of 150 volunteers wereselected as test subjects on whomPRP extraction was performed. Thosewith platelet count less than two lakhsand suffering from infective orconnective tissue pathology wereexcluded from study. This study wasapproved by institutional ethicscommittee and informed consent takenfrom all volunteers. Two spin techniquewas used to extract PRP.

Equipment required for processing40ml venous blood (figure1)

1. Centrifuge (Remi R-4C),4000rpm with countdown timer fixedangle rotor, capacity 4x50 ml

2. First centrifugation (soft spin) :2x20cc syringe, 6ml ACD-A (Acidcitrate dextrose), 2xBD insulin syringe

3. Second centrifugation (hardspin): 20cc syringe, 3-way cannula, BDinsulin syringe

4. PRP extraction: 10cc syringe,3-way cannula.

Platelet Rich Plasma extraction with low cost assembled kitDr. Amarnath Surath, Dr. Rami Reddy Mettu & Dr. N. Suresh Prasad Reddy

38 Orthobiologics

Figure 1:Consumables required for PRP

extraction.

Procedure

Step I :

Three ml ACD-A was taken in 20mlsyringe and 17 ml blood drawn fromthe ante cubital vein. Blood (0.3 ml)was sent for initial platelet

count.(figure2) Nozzle of the syringewas blocked firmly with the cap of theinsulin syringe. With a heavy scissorsflanges on the either side of the syringebarrel were cut.( Figure 3) The pistonwas cut leaving one inch projecting outof the barrel. The two syringes wereplaced in centrifugation tubes oppositeto each other with the tip pointingupwards. This will help balance thecentrifuge and minimize vibration.(Figure 4) The first spin was set at1500rpm for 15mins without brake(Soft spin).

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Figure 2: 20cc syringe with 3ml ofACD-A and 17ml whole blood.

Figure 3: Modifying syringe to fit intocentrifuge rotor.

Figure 4: Lab centrifuge.

Step II

After soft spin blood in the syringewas separated into three layers. Thebottom layer comprises of RBC, themiddle layer leucocytes and the toplayer, plasma with platelets insuspension. (figure 5) The serum wasdrawn into the second 20cc syringethrough the 3-way cannula. Care wastaken to avoid aspiration of the buffy

coat and RBC.( Figure 6) Thecombined volume of plasma from bothsyringes ranged between 16-18ml. Thesyringe was again capped andmodified to fit the centrifugation tube.A counter weight having an equalvolume of saline was placed in theopposite tube for balance. ( Figure 7)The second spin was set at 3500rpmfor 7minutes (Hard spin).

Platelet Rich Plasma extraction with low cost assembled kit

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Step III

At the end of second spin the syringe contains Platelet Poor Plasma (PPP)on top and platelets along with scanty WBC at the bottom in the form of a pellet.(Figure 8) The supernatant plasma is drawn and discarded leaving about fourml at the bottom which is reconstituted to form PRP fraction. This is sent forculture and counts.

Figure 7: Syringe prepared for secondspin along with counter weight.

Figure 5: Appearance after soft spin[1500RPM for 15 minutes].

Figure 6: Aspirating serum intosecond syringe after soft spin.

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Results

PRP was extracted from 150samples and the results are tabulatedin Table 1. The Average Platelet countfrom peripheral venous blood was 259x 103 /µl (range from 211x103 to

427x103/ µl). Average platelet countsafter concentration were 1743x103 / µl(range 810x103 to 2700x103/ µl).(chart1). Only three patients had valvesbelow one million which was due toblood leak from the syringe duringcentrifugation. Platelet counts in

Figure 8: Appearance of syringe aftersecond spin.

Figure 9: Typical blood counts from peripheral blood and PRP sample showing baseline platelet count of 2.35 lacs increased to 15.45 lacs after concentration.


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excess of 1million/µL were obtained in147 (98 %) which validates themethodology. The amplification ofplatelet count ranged from 5-7x. All thesamples were sent for culture andsensitivity studies and none of them

showed bacterial contamination. Thisvalidates the technique as sterile andsafe for clinical application. Staticallyanalysis shown in table 2 proved tohighly significant.

Table 1: Showing platelet counts before and after PRP extraction.

2 to 2.5 lakhs 98

2.5to 3 lakhs 21

3 to 3.5 lakhs 14

3.5 to 4 lakhs 10

4to 4.5 lakhs 7

6 to 10 lakhs 5

10 to 12 lakhs 34

12to 15lakhs 17

15 lakhs and above 93

Pherpherial bloodplatelet count

No of persons Platelet count inPRP extract

No of persons

DiscussionA number of devices are available

in the market which extract PRPutilizing single or double spintechniques. Whatever the equipmentused, the concentrations of plateletsshould be at least 1,000,000/µl in orderto have therapeutic effect. High costof equipment has deterred mostsurgeons from using the kits frequently.This study made use of readilyavailable consumables which aresterile and pyrogen free. We avoidedthe use of centrifugation tubes as theyare meant for laboratory use and mightnot be pyrogen free. This is a low cost

kit compared to commercially availableequipment [ Rs:350]. All samples weresent for culture and sensitivity studiesand returned sterile. Potentialcontamination was avoided as it wasa closed loop procedure performed atpoint-of-care. The choice ofanticoagulant was between heparinand ACD-A. Initial trials were done withheparin which failed to return adequateplatelet counts hence we shifted toACD-A. Slichter and Harker stated thatACD-A maintains intra plateletsignaling thus improving theresponsiveness of platelets. Venousblood was drawn with a 21G

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hypodermic or butterfly needle toprevent inadvertent activation ofplatelets. The butterfly needle wasused when more than one syringe ofperipheral blood was needed. The useof ACD-A resulted in better plateletcounts but no attempt was made to testtheir viability. Platelet counts wereperformed on the peripheral bloodsample and the PRP sampleemploying automated cell counter(Sysmex KX-21N). We did notmanually count the platelets althoughpublished studies mention that thevariation between automated andmanual counting was only 1.1%. ThePRP was leucocyte depleted becausemeticulous care was taken to avoid thebuffy coat after the first spin. Schneiderand Tiidus have stated that thepresence of leucocytes mightexacerbate existing tissue damage.A peripheral blood volume of 20-40mlproduced 2-4ml of PRP which is readyfor injection. Activation of the plateletconcentrate is beyond the scope of thisstudy as it describes only platelet

extraction procedure and not clinicalapplications. Calcium chloride addedto PRP in a ratio of 1:10 is a potentactivator, however the presence ofcollagen as in tendinopathy anddiabetic wounds serves to activateplatelets. Once activated 95% growthfactors are released within one hourbut secretion continues through thelifespan of the platelet. Fukaya et alhave used similar methodology andachieved platelet counts in excess ofsix million. This study was performedon two volunteers only and utilizedeight syringes which increases thechances of contamination. Manypublished articles state that very highplatelet counts may have an inhibitoryeffect, hence the question, “How muchis too much?”, remains unanswered.One limiting factor in this study was thatno attempt was made to quantify theindividual growth factors. This kit willfacilitate the design of randomizedcontrol trials to validate the indicationsfor platelet use in a variety of musculoskeletal conditions.

ConclusionThis technique of PRP preparation consistently gives the optimal platelet

concentration. It can be used as a core technique for the design of RCT’sto evaluate the rationale for using platelets in musculo skeletal conditions.Aseptic technique is assured as this is a closed method of extraction.


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45

Introduction

Platelet-rich plasma (PRP) is anemerging, attractive and promisingtherapy for a number ofmusculoskeletal disorders. Althoughthe efficacy of it‘s use in orthopaedicclinical practice has been highlydebated, the safety of its use has beenwell-evidenced. The use of PRP intendinopathies has been gainingpopularity among the orthopaedic andsports medicine specialists. Of late,there has been increasing use of PRPparticularly in common tendondisorders such as tennis elbow, plantarfasciitis and Achilles tendinopathy. It isknown that in the general population,the lifetime cumulative incidence ofAchilles tendinopathy is 5.9 % amongsedentary people and a staggering 50% among elite endurance athletes andit is also estimated that plantar fasciitishas an incidence of 4 to 22 % amongthe population of runners. Patients

Platelet-rich plasma therapy in tendinopathiesDr Vijay Shetty, Dr Saurabh Talekar & Dr Aditya Kaja

suffering from these tendinopathiesoften require lengthy rehabilitation oreven surgical intervention, ultimatelyretiring from athletic activities. Forthese reasons, the use of PRP intendon disorders has gained popularityin recent times. This article aims to lookat the currently available evidence onthe safety and efficacy of the use ofPRP in orthopaedic clinical practice.

The basic mechanism of action

Platelets are normal componentsof the blood. During a typical normalwound healing, platelets migrate to thearea along with other cells(leukocytes). The platelets areactivated, and they degranulate torelease what are called growth factors.These growth factors play a role instabilising the wound and begin thehealing process. Platelet-derivedgrowth factor (PDGF), peaking shortlyafter tissue damage, plays a centralrole in the healing process.

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The solution in question

PRP is the plasma fraction ofblood with a platelet count roughly 4to 5 times above the baseline. Thereare various methods of PRPpreparation and this day-and-age,there are many ready-to-use kitsavailable commercially. When itcomes to the treatment oftendinopathy, there is still muchdebate, on an ideal PRP preparation.There is debate about the type ofpreparation, debate about whetherfresh or frozen PRP preferred, andthere is debate about leucocyte-rich(LR-PRP) or leucocyte-poor PRP (LP-PRP). Researchers from Francestudied five different types of PRPpreparations from a single donor andnoticed significant variations in theprepared solution and postulated thatthis may be the reason for thevariability of results in PRP studies.Again, there is enormous confusionabout fresh PRP versus freeze thawedPRP.

Simply put, the current PRPpreparations are either LR-PRP or LP-PRP. Our current thinking is LR-PRPis pro-inflammatory, and LP-PRP isanti-inflammatory.

The evidence

The use of PRP in the treatmentof tendinopathies has been a subjectof enormous debate. Table 1 showssome of the studies that have shownvariable results with the use of PRP indifferent tendon indications.

Currently, high-quality evidence isavailable to support the use of LR-PRPfor tennis elbow, moderate high-qualityevidence is available to support the useof LR-PRP in patellar tendinopathy.PRP injection has also proven to besuperior to corticosteroid injections inrecalcitrant plantar fasciitis However,there is not enough evidence even atthis point to prove whether PRPtherapy is an effective method oftreatment for Achilles tendinopathy.

47Platelet-rich plasma therapy in tendinopathies

48 Orthobiologics

The conclusion

It appears, from the available literature, that PRP is a safealternative compared to other therapies in musculoskeletal tendondisorders. However, there is mixed evidence on the efficacy of thisprocedure on specific tendon problems (indications). There is a needfor research comparing various types of PRP and Autologous bloodinjections and its efficacy in specific indications. Based on the currentlyavailable literature, LR-PRP appears to be an attractive option for tenniselbow, plantar fasciitis and patellar tendinopathies. Efficacy of PRP onAchilles tendon and rotator cuff is poor. Given that the use of PRP issafe, it is recommended that clinicians should take fully informed consentand discuss the pros and cons of this therapy and then offer thistreatment to informed patients.

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Osteoarthritis (OA) of the knee isone of the main causes ofmusculoskeletal disability. Autologousplatelet-rich plasma (PRP), whichcontains a pool of growth factors,appears to offer an easy solution fordelivering multiple growth factorsneeded for tissue repair. Today, PRPis being portrayed as a ‘‘wonder drug,’’without sufficient evidence to supportits application. Most reports of PRP useare anecdotal and stem frominsufficient evidence. The procedure isunder a cloud of skepticism as it is notapproved by FDA for use inosteoarthritis of the knee. Before totallydisregarding PRP one must keep inmind that powerful industrial influencesmay be at play as this modality hasthe potential to reduce the number ofinterventions ( TKRs ) which mightadversely affect their balance sheets.We have reviewed three studies ofwhich two draw from personalexperience and arrived at a broadguidelines for those interested inpracticing PRP therapy.

PRP in OA Knee : Consensus Document

Study #1

Treatment With Platelet-RichPlasma Is More Effective ThanPlacebo for Knee Osteoarthritis : AProspective, Double - Blind,Randomized Trial Sandeep Patel,*MS, Mandeep S. Dhillon,*y MS,FAMS, Sameer Aggarwal,* NeelamMarwaha,z MD, FAMS, and AshishJain, MD "The American Journal ofSports Medicine, Vol.41, No.2, 2013."

Sandeep Patel et al in their studysupport the short-term effectiveness ofPRP injection over a placebo forrelieving pain and stiffness andimproving knee functions in early kneeOA. Benefits in early OA are morepronounced and a single dose of PRPis as effective as a double dose. Theeffect tends to taper off over time,leaving open the option of stagedinjections over many months as apotential future therapeutic regimen.

Study #2

Role of double vs triple shotintra articular platelet rich plasmain early osteoarthrosis of kneeDr. K Satya Kumar, Dr.Riyaz Babu

Dr. Koduru Satya Kumar

50 Orthobiologics

Shaik, Dr.Lakshmi NarayanaPaladugu and Dr.Amarnath SurathInternational Journal ofOrthopaedics Sciences 2018; 4(2):48-50

This is a prospective study inwhich 120 patients were involved. All120 were of Kellegren Lawrencegrade-II. Forty five of them were maleand 75 of them were female. A total of135 knees were involved, half thepatients were given two shots of intraarticular injection of PRP along withpost procedural oral analgesics for 1week and the other half were giventhree shots of intra articular injectionof PRP, with topical analgesia withoutoral analgesics. Results were analysedusing pre and post procedure VAS painscale grading and patient questionaire.

Conclusion

As already known platelet richplasma induces tissue healing and hasgood regenerative capacity which isthe main basis for its usage inosteoarthritis. I conclude in my studythat for KL-GRADE2 (A1&B1), earlyosteoarthrosis of knee two shots ofintra articular injection of platelet richplasma with minimal use of analgesicsis enough to show short term goodresults. Very few patients needed a

third injection of PRP. For KL-GRADE2(A2 & B2) early osteoarthrosis of kneethree shots of intra articular PRPinjection, with topical analgesia withoutoral analgesics showed excellent longterm results. This study helped us inselecting the number of PRP injectionsto be used in KL grade 2 OA. Patientswho received 3 shots of PRP showedexcellent long term results without useof oral analgesics. Its efficacy in morethan 2 years follow up to be studiedfurther.

Study #3

Dr Amarnath Surath and Dr Pavan Kumar

Telephonic interview of patientswho received PRP injections for KLGrade-I & II OA knee between 2016-18. The protocol was to give threeinjections at two week intervals. Thevolume of PRP taken was 3ml whichgave platelet counts in the range of10-18 lacs. Injection was given with theknee flexed 90 degrees to one side ofthe patellar ligament. Patient wasadvised quadriceps exercises astolerated. A total of 106 patients werecontacted over telephone andanswered a questionnaire regardingresponse to PRP therapy. Twentyseven of them could not be contacted

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and were lost to follow up. Sixtypatients were between ages 35-55years and 18 more than 55. Malefemale ratio was predominantly female( M19 : F 41 ).

Results : Twelve patients who hada single injection and dropped out ofthe study. Among the remainingpatients VAS improved around 50% in40 patients and 50-75% improvement

in eight patients. All 48 patients werenot on NSAIDs and able to do theirADL. Patients aged above 55 yearshad a different story to tell. Six patientshad no relief at all, and 12 hadimproved VAS scores. In all patientsthere was reduction in pain but theyneeded NSAIDs occasionally. Therewas no significant improvement inwalking distance or stair climbing.

The Consensus

KL Grade I & II are ideal for PRP injection.

Varus & Valgus deformity should be ruled out with Scanogram.

Optimal injection sequence is 3ml x 3 times at 2week intervals.

PRP injection must be followed by Knee ROM and Quads exercises.

PRP in OA Knee : Consensus Document

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53

In adult skeleton there are twotypes of bone marrow which are redmarrow and yellow marrow. Yellowmarrow is filled with adipose tissue andis inactive by function. Red marrowpossesses hematopoietic cells andmesenchymal stem cells.Mesenchymal stem cells (MSC) havethe capability to differentiate into celllines like cartilage, bone, tendon,muscle and nerves. Benefits of BMAcare achieved either by proliferation anddifferentiation into variety of celllineages or by elution of growth factorsand cytokines to hasten the processof healing in tissues with attenuatedhealing potential.

Components of BMA

In normal bone marrow aspirate(BMA),common components presentare erythrocytes(22-28%), neutrophils( 3 2 - 3 7 % ) , l y m p h o c y t e s ( 1 3 % ) ,eosinophils (2.2%), blast cells(1.4%),monocytes (1.3), basophils(0.1%) andmegakaryocytes in variable

percentages. Apart from the abovecomponents, BMA containsmesenchymal stem cells and variousgrowth factors. An adult bone marrowhas about 0.001% to 0.01% ofmesenchymal stem cells in 7-30 cellsper million nucleated cells.Growthfactors like PDGF, TGF-â, BMP2and7 and IL-1RA are also present in theBMA in variable concentrations(Table-1).

Main aim of bone marrowconcentration is to increase the cellcount of mesenchymal stem cells.Theconcentrations of MSC in bone marrowalone is relatively low, hence theaspirate is concentrated bycentrifugation in order to increase thepercentage of MSCs. Cell count inbone marrow aspirate is 612±134 percm3 compared to bone marrowaspirate concentrate is 2579±1121 percm3 which is a fivefold increase in thenumber of mono nuclear cells (Figure1).

Bone Marrow Aspirate Concentrate (BMAc)Dr. A.V. Dakshina Murthy

54 Orthobiologics

Figure 1 : Histopathological picture showing mesenchymal stem cells in bonemarrow aspirate with admixture of RBC. (A), mesenchymal stem cells in BMAc

extracted by density gradient centrifugation (B).

A B

Table-1: Growth factors present in mesenchymal stem cells

PDGF Stimulates cell proliferationChemotaxisStimulates angiogenesis

TGF-β Stimulates production of collagen type I and type IIIAngiogenesisRe-epithelializationPrevents collagen break down

BMP2 TGF-β signaling pathwayHedge hog pathwayCytokine and cytokine receptor interactions

BMP7 Phosphorylation of SMAD1 and SMAD5- inducetranscription of numerous osteogenic genes.

IL-1RA Pain relief

GROWTHFACTORS FUNCTIONS

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Bone marrow Aspiration

Commonest source of bonemarrow aspirationis from iliac crest,tibia or calcaneum. Among all of thesources,iliac crest yields highestconcentration of mesenchymal stemcells. Graft is harvested bysector rulein iliac crest, which roughly divides theentire iliac crest into six zones. Of thesesix zonesthe posteriorcrest is preferredsite as it contains higher concentrationof MSCs (higher by 1.6 timescompared to anterior iliac crest).

Concentrating the bone marrowwill eliminate non- nucleated cells likered blood cells and increase in thenumber of mesenchymal cells.

Protocol for BMAc FICOLL densitygradient centrifugation

Aspirate bone marrow intoheparinized10ml syringe.

After aspirating 1.5 to 2ml ofmarrow the needle must be withdrawnby a few millimeters and rotated 90degrees to reduce the admixture ofperipheral blood. The number ofaspirations per site should not exceed6-8, after which the needle placementin bone should be changed.(Figure 2)

Figure 2 : Aspiration of Bone marrowfrom anterior iliac crest.

Collect into 50 ml centrifugationtube with 8ml heparin.

Take 25 ml Ficoll in four 50mlconical bottom centrifugation tubes.

Layer 25 ml of marrow on top withtube tipped at 45 degrees. (Figure 3)

Figure 3 : Layering of bone marrowaspirate over ficoll solution.

Bone Marrow Aspirate Concentrate (BMAc)

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Figure 4: Tubes before centrifugation. Figure 5 :Concentrate obtained aftercentrifugation.

Centrifuge at 2500 rpm for 40 minutes.

Aspirate the smoky layer (arrows) at the interface between plasma and ficoll. (Figure 5)

Add 3 volumes of Phosphate buffered saline to the cells, centrifuge@2000 rpm for 10 minutes.

Repeat and wash again

Resuspend and use. Approximate yield of BMAc from 100ml of marrow is 5-7ml. (Figure 6)

Figure 6: Mesenchymal cells pellet.

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Identification of Stem cells

The mere presence of mononuclear cells does not confirm the presenceof mesenchymal stem cells. CD34 marker is used to identify stem cells. (fig.7)

The concentration of mononuclear cells was measured using an automtedcell counter and increase in cells was documented (fig.8)

Marrow Counts BMAc Counts


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Complications

In literature very few complications during harvesting and administrationare described with low incidents in table 2

Table: 2 complications during procedure

During harvesting During administration

Hemorrhage infection

Neurovascular injury Pulmonary embolism and respiratorycomplications

Pathological fracture tumor genesis

Clinical applications :

Non unionBone defects

Hernigou et al. used Bone MarrowAspirate Concentrate in the treatmentof atrophic non-union in 60 patients. Apositive association between thequantity of hard callus and the number(p = 0.04) and concentration (p = 0.01)of fibroblast colony forming (FCF) unitsin the graft was reported by them. Inthe seven non-united tibias, theconcentration (p = 0.001) and the totalnumber (p < 0.01) of progenitors cellsinjected were signicantly lower than inthose that have united. One morefinding they made was in the timeinterval needed to achieve union. This

was negatively correlated with the FCFunits’ concentration at the site of thegraft (p = 0.04).

Avascular necrosis Studies were conducted on

management AVN of the femoral headAVN using BMAC injection

Of all Hernigou and Beaujeanwere the first to describe the protocolfor this . They combined BMACinjection with conventional coredecompression (CD). BMAC wasinserted into the necrotic area withinthe femoral head.

189 hips were studied with thisprocedure.

Their results were….excellentoutcomes in 93% hips at the pre-

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collapse stage, with only six percentcases required total hip replacement(THR) later

ArthrodesisBenign bone lesionsChondral defects

Giannini et al tried the use ofBMAC as a single-step technique forthe reconstruction of cartilage defectsof the talus, comparing this procedureto open or arthroscopic autologouschondrocyte implantation (ACI)

In the above study of 81 patientsthe mean American Orthopaedic Footand Ankle Society (AOFAS) scoreimproved signicantly (p < 0.0005) atan average of 59.5 ± 26.5 months.There were no signicant differences inthe change of AOFAS scores betweenthe all groups. Histological evaluationsemphasised the formation of type IIcollagen and proteoglycan expression.However, BMAC provided theadvantage of permitting a noticeabledecrease in morbidity as a ‘‘one-step’’technique

Spinal fusionOsteoarthritis - In osteoarthritis,

as per literature BMAc is more effectivein Kellgren-Lawerance grade 2

changes than grade 4 changes. Twoto six injections at an interval of two tothree months is advised for excellentresults. Main complications are painand swelling for six to eight weeks.Hence BMAC can be recommendedfor early stage of osteoarthritis.

Tendon injury

A prospective multicentre studywas done by Centeno et al This studycompared the results of use of BMACfor the treatment of osteoarthritis (OA)shoulder having rotator cuff pathologyand the use of BMAC for the treatmentof osteoarthritis without any rotator cuffpathology.

A total of 115 shoulders weretreated with BMAC injection principallyfor glenohumeral OA . The studyincludes both categories of cases, i.eglen humeral OA with rotator cuff tearsand without a rotator cuff tears

The mean disabilities of the arm,shoulder and hand score and VASimproved signicantly from 36.1 to 17.1(p < 0.001) and 4.3to 2.4 (p < 0.001),respectively. These results wereconsistent with a mean subjectiveimprovement of 48.8%. They reported


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no signicant adverse events at twoyears follow-up post surgery

Advantages of BMAC areOn site procedureNo amplification or manipulation

of cellsNo risk of disease transmission

Low risk of infectionDay care procedure

Disadvantages

Pain due to local anesthesia

Variable quantity of cells

Side effect of erythrocytes

Keypoints :

BMAc is a rich source of haematopoietic stem cells.

Ficoll separation is “gold standard”.

Performed in operatioon theatre with low logistical requirement& cost.

Will hasten the process of healing in tissues with attenuatedhealing potential.

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Bone union is the desired endpointafter any fracture. Many factorsinfluence fracture healing in the formof patient selection, implants andsurgical technique. Three componentsare needed for fracture to progress tohealing, namely the presence of stemcells, growth factors, and a biologicscaffold are integral to this process.Bone marrow aspirate (BMA) has beenutilized as a source of bone marrow-derived mesenchymal stem cells (BM-MSC) with its relative ease of harvest,low morbidity, and feasible cost. Bonemarrow contains low percentage ofmesenchymal stemcells( MSCs), .001-.01% of nucleated cells. Typically theaspirate is concentrated bycentrifugation to increase the ratio ofMSCs. Concentrated bone marrowaspirate(cBMA) provides both stemcells and growth factors and relies onthe host tissue to provide scaffold.BMAC has proved itself as a valuableadjunct in treatment of fractures,cartilaginous defects and tendoninjuries. This chapter will confine itselfto its role in fracture healing. Evidence

BMAc in Nonunion of Fractures

suggests that stem cells act to directlocal cells to stimulate regenerationand repair that is specific to eachtissue. This process is mediated bysecretomes from the stem

cells, which allow their adaptationin each environment and thereforeprovides the appropriate growth factorsand cytokines necessary to stimulateeach tissue in a different fashion.

Bone consolidation and time tobone union was improved in patientsreceiving BMAC, with faster healingrates when compared to patients in theautograft group. One study found asignificantly lower number of progenitorcells in patients who did not achieveunion. Time needed to obtain unioncorrelated directly to the concentrationof colony forming units in the graft.Lastly, one study evaluated the efficacyof BMAc in the treatment of open tibiafractures and found adequate boneconsolidation and bone callusformation in all patients. BMACapplication was used in combinationwith Demineralised Bone Matrix/

Dr. Amarnath Surath

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rhBMP-2, freeze-dried allograft, orcancellous bone chips. Presentexperience is with direct injection intofreshened fracture site with or withoutaddition of fibrin sealant (Tiseel).Injection of BMAC into the site ofnonunion was accomplished byfluoroscopic visualization.

BMAC isolation was by densitygradient centrifugation with Ficoll asdescribed in the previous chapter. Theresidual ficoll was removed by serialwashes ( two) and 0.3 ml of the samplewas sent for vital staining(Trypan Bluestain), IHC with CD-34 which will stainMSC’s and cell count. Once yield andvitality were established the samplecan be injected into fracture site. Theaddition of fibrin sealant is the choiceof the surgeon but it adds to the costof the procedure.

Inclusion Criteria for BMACinjection for Delayed/Non Union

A diagnosis of nonunion was madeby the clinical examination andradiographic data. Clinical evidence ofa nonunion was determined bydocumented pain and motion at thefracture site. “Nonunion” wasdetermined by a lack of radiographic

evidence of bone bridging on 3 of 4cortices in two planes of X-rays at 6months after injury or a fracture thathad not shown in any progression ofhealing over a three-month period.

Exclusion criteria

Exclusion criteria were presenceof infection clinically or by positiveinflammatory markers, ongoingtreatment with immunosuppressantdrugs including glucocorticoids,chemotherapy or colchicines. Patientswere excluded if they were pregnantor during lactation. Patients withautoimmune deficiency syndrome,hepatitis, or a medical history of alcoholor drug abuse were also excluded.

Assessment of bone healing

All patients were monitored usingthe same protocol during thepostoperative period. Patients werefollowed up in the outpatient clinic for1 month, 3 months, 6 months, 9months, 12 months, 18 months, and24 months after the procedure. Thephysical examination assessed pain,sensation of stability, and

ability to walk with or withoutcrutches. X-rays were taken in two

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standard planes (anteroposterior andlateral) at all visits. Radiographicevaluation was performed via X-rayanalysis. Assessment of new boneformation and remodelling was basedon the modified Lane and Sandhuradiological scoring system. The scorefor bone formation was defined as 0(no new bone formation), 1 (<25% newbone formation), 2 (25–50% new boneformation), 3 (50–75% new boneformation), or 4 (>75% new boneformation). The score of union was 0(full fracture line), 2 (partial fractureline), and 4 (absent fracture line). Theremodeling score was 0 (no evidenceof remodeling), 2 (remodeling of theintramedullary channel), and 4 (fullremodeling of the cortex). Themaximum points could be achievedwas 12. Fracture healing wasassessed by lack of pain during

weight-bearing and bridging ofthree out of four cortices in bothanteroposterior and lateral radiographicviews (Liebergall et al., 2013). Boneunion was established when bothclinical and radiographic evidence

were in agreement (Tressler et al.,2011). Evaluation of the radiographs

as part of the clinical follow-up wasperformed by the non-blindedsurgeons. Every side effects resultingfrom the procedure were assessed andrecorded. Partial weight bearing wasonly allowed after the appearance ofthe bone callus and with signs ofstability upon physical examination.The defined clinical protocolestablished that, if the patient did notpresent signs of bone consolidation sixmonths after the procedure, a secondintervention would be indicated, asituation considered a treatmentfailure.

Procedure

Once diagnosed with non/delayedunion based on clinical and radiologicalfindings the patient was taken up forBMAC injection. First and foremost thepatient and relatives were counseledthat the procedure may not produce thedesired result i.e. bone union and thepossibility of requiring multipleinjections was explained. Underaseptic conditions, in the operatingtheatre, bone marrow was aspiratedand processed as mentioned in theprevious chapter. The average yield ofBMAC from 100ml of bone marrow is

BMAc in Nounion of Fractures

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5-10ml. The patient is suitablyanaesthetized, draped and fracture siteidentified under fluoroscopic guidance.A small stab incision was given andfracture site was freshened with

curettes. The BMAC was then injectedinto fracture site through a differenttrack. Serial radiographs were takenfor follow up.

Case Studies

# Case 1

Male ,23 yrs presented with left proximal tibial fracture following RTA. Xraysshowed grossly comminuted proximal tibia fracture ( Fig 1a ) with swelling ofthe soft tissues and blisters. Fracture was stabilized with Ender nails and POPslab ( Fig 1b ).

Three weeks following fixation BMAC injection was carried out into thefracture site under fluoroscopic guidance. No attempt was made to freshen thefracture site as it would disturb the scaffold formed in the interval between thefracture fragments. Complete consolidation was observed at 20 weeks(Fig 1c).

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# Case 2

Male, 80 sustained comminuted sub trochanteric fracture of the right femurdue to fall from stairs ( Fig 2a ). DHS fixation was carried out but due to extensivecomminution biological fixation was achieved ( Fig 2b ). At six weeks BMACwas injected in between the fragments and the fracture consolidated in twentyweeks. The follow up Xray at 6 months shows stable bone healing and patientwas ambulatory without support (Fig 2c).

# Case 3

Male, 21 sustained pathological fracture of the left distal femur secondaryto chronic osteomyelitis (Fig 3a). Antibiotic PMMA coated nail was inserted tostabilise the fracture. Infection was controlled but fracture showed evidence ofdelayed union (Fig 3b ). He received two injections of BMAC at 8wks and 12wks.The fracture consolidated by 24 weeks and resulted in bone union as well asinfection control (Fig 3c).


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# Case 4

34 year old male sustained open fracture mid shaft femur in RTA. He underwentORIF and primary bone grafting after thorough debridement and lavage. BMACwas injected into the bone graft at six weeks and fracture consolidated by 20weeks ( Fig 4 ).

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Conclusion

BMAC consistently results in fracture healing and has a definiterole as an adjuvant. It precludes the need for cancellous bone graftingwith the resultant morbidity of an additional surgical intervention. Itcan be performed as a day care procedure.


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69

Mesenchymal Stem Cells in Management of AvascularNecrosis Femur Head

Dr. Ramesh K Sen

Osteonecrosis (Avascularnecrosis) of the femoral head (ONFH)is a disorder with a spectrum ofdifferent etiologies. Exact pathogenesisis still not clear. The clinicalpresentation is variable with somepatients having significant symptomsin early phase of the disease while inothers, it may not manifest clinically tillfemur head is collapsed. Among manyetiological factors, steroids and alcoholremain the common causes, but inmany patients, no clear reason can bedefined. While x rays may not help inthe initial stage of lesion, MRI is usuallythe diagnostic investigation. As it runsa slow course in most patients, aconservative approach of non-weightbearing has been advocated. Therehave been a lot of interest inmedical management especiallybisphosphonates, but still universalacceptance is lacking. Most orthopedicsurgeons carry the perceptions thattotal hip arthroplasty is the final andan essential procedure in mostpatients.

At an early stage of presentation,common surgical intervention hasbeen the core decompression, with thegoal of delaying or preventing the needfor THA. Even though this procedurehas been used for many years, itsefficacy has remained controversialwith no consensus in literature. Oneof the reasons proposed for its failurewas lack of induction of any osteogenicactivity in the necrotic area. Thedevelopment of regenerative medicinehas now gone beyond this limit and inrecent years core decompression hasbeen supplemented with additionalprocedures like an osteoinductiveagents that can enhance bone repair.

Stem cells are a group of cells withthe ability to self-renew and formdifferentiated cells. These cells playimportant roles in development anddisease. Adult stem cells, whichinclude mesenchymal stem cells(MSCs), have been reported as apromising approach for theregeneration of various tissues. MSCs

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were first described in human bonemarrow and called bone marrow stemcells (BMSCs). These cells howevercan be isolated from many othersources also, including adipose tissue,the synovial membrane and theumbilical cord etc.

Hernigou et al were the first toreport in 2002, that thesemesenchymal Stem Cells can improvesurgical outcomes of CD technique inavascular necrosis femur head. Theyhypothesized that there is aninsufficient supply of progenitor cellsenhancing bone remodeling in areasof AVN in patients with ONFH. TheMSCs are instilled into the necroticarea after core decompression. Theyalso proposed that thesenonhematopoietic progenitor cellsdifferentiate in osteoblasts under theinfluence of growth factors such asbone morphogenetic proteins, platelet-derived growth factor (PDGF),transforming growth factor β (TGF-β),insulin like growth factor, fibroblastgrowth factor (FGF), and parathyroidhormone. Thus combination of MSCstransplantation and CD surgery canenhance femoral head repairpromoting reconstruction and

creeping substitution of new bone.Hernigou et al. publishes a clinicalstudy using core decompressionand autologous bone marrowtransplantation for the treatment of 189hips in 116 ONFH cases. Only 9 casesof 145 hips, operated during Ficatstages I-II, required hip replacementafter a mean 7-year follow-up, whereas25 of 44 hips treated during Ficat stagesIII-IV, needed joint arthroplasty.Subsequently Gangji et al in aprospective, randomized, double blindtrial, compared the surgical outcome of2 groups of patients treated withisolated CD or CD and implantation ofautologous bone marrow cells. In 24cases at ARCO Stage I and II, theyobserved a significant improvement ofpain and lower rate of radiographicprogression in the group treated withthe combined approach. However, theydid not report a significant difference inboth the groups in terms of subsequentTHA.

Many clinical studies have nowappeared in literature where thetherapeutic effect of stem cells onONFH have been evaluated. Majorityof authors demonstrated positive clinicoutcomes, including reduced pain,

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improved function and motion, delayedprogression or the avoidance of THA.However, few others like Pepke et al.did not report any significant benefitfrom the additional injection ofconcentrated bone marrow aspiratecompared with the effects of CD alonein the short term. Some retrospectivecomparative studies also drew similarconclusions.

A recent meta-analysis howevershowed the usefulness of implantationof autologous MSCs into the CD track,particularly in the early (precollapse)stages of ONFH, and concluded thatcombined use of CD with MSCinstillation could improve thesurvivorship of femur heads andreduce the need for hip arthroplasty.Another meta-analysis having eightrandomized controlled trials alsodemonstrated the benefit of thecombination of CD with regenerativetechniques as compared with CDalone, in providing a significantimprovement in survivorship over thetime.

There can be controversies due toheterogeneity among studies,including differences in patient

selection, cell harvesting, cellprocessing, and cell delivery etc. butoverall, it seems that the generaloutcomes of the use of stem cells totreat ONFH have been good.

Some studies have looked into thefactors which may affect the outcomeof treatment in using MSC in AVN. Mostunderstood factor in usage of stem celltherapy is the stage of ONFH asreported by Ma et al. Hauzeur et al.also looked into the delayed cases andreported that in stage III ONFH, theimplantation of bone marrow aspirateconcentrate (BMAC) after CD did notproduce any improvement. The stageIII and stage IV patients may not bethe candidates for this therapy whileearly-stage (stage I or II) patients willbe a more appropriate choice. Inaddition, authors have observedreported that patients withposttraumatic osteonecrotic hips haddifferent outcomes than did patientswith non-traumatic hips, suggestingthat etiology also affects clinicaloutcomes. It has also been very welldocumented that patients with a lowmodified Kerboul grade achieve betterresults. It seems that the stage, size,morphology and even etiology of

Mesenchymal Stem Cells in Management of Avascular Necrosis Femur Head

72 Orthobiologics

ONFH may be important factorsassociated with the treatmentoutcome. Thus, patient selection iscritical in treatment outcome in usageof stem cell therapy in ONFH.

It has also been seen that agingis a factor in decreased number ofMSCs isolated from a donor and theproliferation ability of those cells.Stenderup et al. have also reportedthat although MSC function wasdecreased in cells isolated from olderdonors in vitro, this difference mightnot affect the ability of the cells todifferentiate in vivo. The authorsconcluded that MSCs isolated fromolder donors maintained normalcellular function but showed aproliferative defect. While Aksu et al.observed that sex may affect thedifferentiation potential of humanadipose-derived stem cells, Sen et al.did not observe sex differences, sideof involvement, and opposite sideinvolvement, having any effect on theoutcomes.

Various types of MSCs have beenused to treat ONFH, including bonemarrow-derived MSCs, adipose-derived MSCs and peripheral bloodMSCs. Among these BMMSCs are

the most commonly used type. Mostlyused as bone marrow concentrate(BMC) or after culture to augment thecell population. Rastogi et al. comparedisolated mononuclear cells withunprocessed bone marrow instillationsand observed the improvements in hipfunction, as measured by the Harris hipscore, in both groups. There was adecrease in the lesion size in theprocessed isolated mononuclear cellgroup, and 3 of 30 hips in theunprocessed bone marrow injectiongroup required total hip replacement. Itseems that the more effectiveprocedure had better outcomes than didunprocessed bone marrow injection forthe treatment of ONFH.

Adipose tissue derived stem cellsare less expensive but also lessinvasive and painful than that used forbone marrow harvesting. An in vitrostudy demonstrated that adipose-derived MSCs may provide a morerobust growth rate and bonedifferentiation potential than bonemarrow-derived MSCs. Although theresults of these studies seemencouraging, there is a lack of well-designed clinical studies to confirm thisopinion. It has been well documented

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that the osteogenesis and proliferationof MSCs are decreased in alcohol-induced and steroid-induced ONFHpatients. Therefore, the efficacy ofstem cells isolated from these patientsmay not have similar therapeuticeffects. Allogeneic stem cells derivedfrom healthy humans may be anoption in treating ONFH in suchindividuals. There is evidence of low-immunogenicity MSCs, allowing theMSCs to be transplanted betweenHLA-incompatible individuals. Humanumblical cord mesenchymal stem cells(hUCMSCs) collection is easy, ethicallyfeasible, yield of UCMSCs is high, andthe cells have low immunogenicity.UCMSCs are also easy to separateand can be amplified in vitro; placentalUCMSCs can typically be grown inculture for 30–40 generations, whileadult BMMSCs can grow only 6–10generations with the sameperformance. Cai et al. performed theco transplantation of autologousBMMSCs and allogeneic UCMSCs fortreating ONFH and observedtherapeutic effects without severeadverse effects at 12 months aftertransplantation. Chen et al. analyzedthe clinical effects of transplanting

allogeneic hUCMSCs for the treatmentof ONFH and observed clear resultswith no obvious side-effects after a3year follow-up. Studies with largernumbers of patients and longer follow-up times are needed to further evaluatethe efficiency and safety of the use ofallogeneic hUCMSCs in treatingONFH.

The prevalence of connectivetissue progenitors in the bone marrowin the iliac crests of patients was aboutone per 30,000 nucleated cells.Hernigou et al. reported that accordingto the mean nucleated cell count perml (18 Ã— 106 cells), the bone marrowharvested from the iliac crest byaspiration contained an average ofapproximately 600 progenitors per ml[38]. If expansion is performed in vitro,more cells will be harvested. It isexpected to have good outcomes withhigh nucleated cell counts [20, 23] butthe optimum number of cells forinjection remains unknown. Based ona mean bone matrix of 33% incancellous bone, it has been estimatedthat there will be about 20 millionosteoblasts or osteocytes per cm3 ofnew bone [39]. Thus, approximately 3× 108 (20 million cells/cm3 ×15 cm3)


74 Orthobiologics

osteoblasts or osteocytes are neededfor new bone repair.

Achieving an objective number ofosteoblasts or osteocytes depend onhow many times the stem cells canproliferate and how many cells caneffectively differentiate into osteoblastsor osteocytes, especially in the usuallyischemic and anoxic microenvironment of the necrotic area of thefemoral head. On the other hand,whether the injection of more stemcells is better and whether there is asafe threshold for the maximuminjection of stem cells remainunknown.

Based on current reportedstudies, except for patients injectedwith approximately 24 × 103 to 25 ×103 cells in early studies reported byHernigou et al. the number of cellsused in most other studies rangedfrom 106 to 109, and the mostfrequently used number was 108 cells.Thus, based on current data, theinjection of 106 to 109 cells may bereasonable. How-ever, the optimalnumber still needs to be investigated.

Many techniques for cell deliveryhave been described , and such

techniques have been usuallycombined with CD. Other techniquesinclude stem cells in impactionallogeneic bone grafting, auto-iliaccancellous bone grafts, porous tantalumrod implantation procedures, poroustantalum rod implantation combinedwith vascularized iliac grafting,interconnected porous calciumhydroxyapatite (IP-CHA) and porousnanohydroxylapatite.

There has also been attemptsusing the MSCs through arterialinjection in management of AVNFH. Caiet al. transplanted MSCs into the medialcircumflex femoral artery, the lateralcircumflex femoral artery or theobturator artery through digitalsubtraction angiography and observeda therapeutic effect on avascularnecrosis of the femoral head (ANFH)without severe adverse effects. Mao etal. also observed that intra-arterialinfusion of PBMSCs could enhance theefficacy of biomechanical supportduring the treatment of ONFH.However, it is difficult to say thatwhether the topical application or intra-arterial infusion of MSCs is moreeffective.

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Some studies also used localinjection with platelet-rich plasma(PRP), pharmacological treatments,such as intravenous iloprost and oralbisphosphonates, or physical therapy,such as low-intensity pulsed ultra-sound (LIPUS).

One of the major concerns in celltherapy is safety. Stem cells have someproperties similar to cancer cells, i.e.a long lifespan, relative apoptosisresistance, and the ability to replicatefor extended periods of time. Thegrowth regulators and controlmechanisms are similar in both cancerand stem cell maintenance. Thereremains a possibility, that stem cellsmay undergo malignant transformation. It has also been reported that thetransplantation of embryonic stem cells

may increase the risk of teratomaformation. Other concerns, includingimmune rejection and geneticmodification, also limit the clinical useof directly transplanted stem cells forONFH.

After a review of current studiesthat used stem cells in the treatmentof AVNFH, most studies reported nosevere complications. There has beenreport of complications like flushing,mild headache and fever in somestudies. Thus, based on the currentstudies, it seems that the applicationof stem cells for the treatment of ONFHis relatively safe.

To conclude it can be safelyassumed that stem cell therapy in AVNof femur head is a viable option withliterature largely in support of it.


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Dr. Karthik Pingle

Orthobiologics in the treatment of Early Osteoarthirits- a Systematic Review

Introduction

Osteoarthritis (OA) of knee is oneof the most common clinicalpresentation in these days amongelderly especially. In Osteoarthritis, asthere is ageing there is cartilage failuredue to decreased inhibitor productionand also reduced chrondrogenicprogenitor cells to repair the worn outcartilage tissue. Recent researchcontinues to highlight the complexnature of OA, with confirmed or likelyrisk factors including demographiccharacteristics, obesity and dietaryfactors, joint loading and injury, andjoint shape and alignment.

There are a number of non-operative options for the treatment ofearly OA. The routine choices includenon-steroidal anti-inflammatory drugs(NSAIDS), muscle relaxants,physiotherapy, knee braces, life stylemodification, chondroprotective agents(diacerein, glucosamaine, chondroitinsulphate), viscosupplementation i.e.

intra-articular hyalyuronic acid (HA).The efficacy of Orthobiologic agents inthe treatment of early OA has beenreported in the literature recently.

Of late, Orthobiologics haveevolved as one of the prominenttreatment modalities for earlyosteoarthritis (OA) of the knee.Biological therapy also called as“cellular arthroplasty” is evolving as anew paradigm in the management ofOA. Exploiting the healing andrejuvenating properties of body’s owncells for the repair and renewal ofdamaged tissues is the basic cruxbehind Orthobiologic therapy. Repairof damaged cartilage and biologicalrestitution can be possible by thejudicious use of autologous biologicalproducts. In the treatment of OA,Orthobiologics occupy an intermediaryposition between the noninvasiveconservative management at one endand the more invasive surgical optionsat the other end. The innovativebiological options which have been

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successful in the management ofosteoarthritis include Platelet RichPlasma (PRP), Bone MarrowConcentrate (BMAC), AutologousChondrocyte Implantation (ACI).

This review describes the role andtechniques of these orthobiologics inthe management of knee OA alongwith their potential merits and demerits.This review predominantly focuses oncontemporary autologous biologicagents that are being used for theclinical treatment of OA knee along witha comprehensive up-to-date review ofpublished literature.

Platelet Rich Plasma (PRP)

Platelet-Rich Plasma Therapy(PRP) was defined by Arnoczky et alas a sample of autologous blood with

concentrations of platelets in a givenvolume of plasma that is above theconcentration found in whole blood (5).Thus, PRP is autologous blood plasmawith concentrated platelets. Typicalconcentration of platelets in PRP isabout 5-10 times that found in wholeblood.

Platelets contain natural sourcesof growth factors, proteins andcytokines that stimulate the healing ofbone and soft tissues, such as Platelet-derived growth factor (PDGF),Transforming growth factor beta (TGF-ß), Insulin-like growth factor 1 (IGF-1),Insulin-like growth factor 2 (IGF-2),Fibroblast growth factor (FGF),Epidermal growth factor (EGF) andVascular endothelial growth factor(VEGF).

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PRP was first introduced in the1980s for the treatment of cutaneousulcers by Margolis et al. Its use wasexpanded in the 1990s for maxillofacialand plastic surgeries. Its use inorthopaedic surgery began almost adecade ago, and it was initially usedwith bone grafts to augment spinalfusion and fracture healing. Theseindications have now expanded widely,with PRP being used to treattendinopathies and tendon injuries (eg.Lateral epicondylitis or Tennis elbow,Achilles tendinosis, rotator cuff tearsetc.), ligamentous injuries andreconstructive surgery (eg. ACLreconstruction), cartilage injuries,osteoarthritis of the knee and hip,muscle injuries and for boneaugmentation during fusions andsurgeries for non-unions.

Treatment options for early ormild osteoarthritis of the knee includeanalgesics, activity modification andphysiotherapy. Over time, patientsusually become refractory to the initialtreatment regime, hencereconstructive surgery becomes thesubsequent treatment modality.Analgesics only help in reducinginflammation and pain but they areineffective in delaying the diseaseprogression. In contrast to thesetreatment options, PRP can shortenthe time away from activity, reduce thenecessary amount of medication andhelp avoid invasive treatment withlonger recovery times, or speed up therecovery. Thus, PRP injection is aneffective and safe treatment for themanagement of early osteoarthritis anddegenerative chondropathy.

Orthobiologics in the treatment of early OA - Systematic Review

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Mechanism of action of PRP:Platelets stimulate the healing processby the release of growth factorsresponsible for almost all repairprocesses that naturally occur in thebody. Platelets additionally cause stemcells to activate, another naturalhealing element responsible for re-building damaged tissue.Consequently, new cartilage tissue isformed and healing occurs. PRPtherapy accelerates this process bydelivering platelets in a concentration5-10 times that of the normal levels.

PRP also contains fibrinogen,which forms a fibrin scaffold afteractivation and helps in tissue healingby filling up the cartilage defects. Thisfurther augments the biological efficacyof PRP.

Preparation of PRP: Aim is tosequestrate platelets in highconcentrations, enough for achievingtherapeutic benefit, and in a viablestate at the same time, so that theycan actively secrete their GFs in therequired amount to stimulate repair.Precautions to be taken during thisinclude strict aseptic conditions,optimum temperature regulation (5 –35o C) and use of an anticoagulant(ACD-A).

I. Sample Collection: 30 cc ofvenous blood is extracted and 3 cc ofACD-A is added to it.

II. 1st centrifugation at 3200 rpmfor 4 minutes.

III. 2nd centrifugation (with thechamber inverted) at 3300 rpm for 3minutes.

IV: Harvest PRP for use.

1st Cetrifuge 2nd Cetrifuge

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Results with PRP

In 2010, Sampson et al conducteda study of 14 patients with OA knee.They administered 3 intra-articularinjections of PRP 4 weeks apart, andobserved significant improvement inpain and Knee Injury scores at 12months follow-up.

In 2011, Wang-Saegusa et alstudied 312 patients with OA knee andtreated them with 3 injections of PRPintra-articularly at intervals of 2 weeks.They reported significant improvementin the pain, stiffness and function at 6months follow-up.

In 2011, Kon et al conducted arandomized controlled trial (RCT)involving 150 patients with early OA ofthe knee, and compared the clinicaloutcome after intra-articular injectionof PRP, and low and high molecularweight hyaluronic acid. PRP was foundto provide longer and better efficacy inreducing pain and symptoms, thanboth low and high molecular weight HAat 6 months follow up.

In 2012, Spakova et al conductedanother RCT with 120 cases of OAknee, 60 of which were given PRPinjections and the other 60 were given

Advantages of PRP: It is a safe,quick and cost-effective procedure,performed easily in an office setting,in a single-stage. Further, the use ofautologous blood mitigates the risk of

disease transmission or rejectionreaction. Proper selection of patientsfor whom PRP therapy can bebeneficial is important.


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HA injections. They found that the painscore and the WOMAC score werestatistically better in the PRP group at6 months follow up.

In 2012, Sanchez et al conducteda multicenter, double-blinded clinicaltrial to evaluate the safety and efficacyof 3 consecutive weekly intra-articularPRP injections versus HA injections in176 patients. PRP was found to besuperior to HA in reducing knee painin mild to moderate OA.

In 2014, Anitua et al conducted asystematic review of international peerreviewed literature published between2008 and 2013 on the efficacy andsafety of PRP in knee OA. A total of530 patients were included by themfrom 5 different studies, 2 out of whichwere RCTs, 2 were prospective studiesand 1 was a retrospective analysis.They concluded that intra-articularPRP significantly reduced pain andimproved function in patients with mildto moderate knee OA.

In 2016, Dai et al performed ameta-analysis of 10 level-I RCTs thatevaluated the efficacy of PRP ascompared to other modalities for thetreatment of early OA knee. A total of

1069 patients were included in theanalysis, and they found that PRP andHA showed similar functional outcome(WOMAC and IKDC scores) at 6months follow-up. However, at 12months post-injection, PRP wasassociated with significantly better painrelief and functional improvement(WOMAC and IKDC scores).

In 2017, Martini et al studied 25patients with grade I and II OA of theknee, and treated them with a singleintra-articular injection of PRP. At 6months follow-up, they found asignificant improvement in the VAS,WOMAC and Knee Injury scores, withno adverse reactions in any of thepatients.

In 2018, Di Martino et alconducted a double-blind RCT of 192patients with OA of the knee, studyingthe efficacy of intra-articular injectionsof PRP versus HA at 5 years follow-up. They found that a significantreduction in IKDC subjective scoreswas observed in both treatmentgroups, with patients in the PRP grouppresenting significantly higher valuescompared with the baseline values.

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Bone Marrow Aspirate Concentrate(BMAC)

Historically, chondral defects inyoung patients have been treated withtechniques such as microfracture andabrasion chondroplasty. With thesetechniques, the resulting fibrocartilageproduction is rich in type I collagen,which may delay the onset ofosteoarthritis, but it is structurallyinferior to the articular hyaline cartilage.Arthroplasty options are available tothose patients who develop end-stageosteoarthritis. Thus, there is a cleardemand for regenerative techniques toslow or even reverse this diseaseprocess.

Mesenchymal stem cells(MSCs) from bone marrow play acritical role in osteochondral repair. Abone marrow clot forms within thecartilage defect either as a result ofmarrow stimulation or during thecourse of the spontaneous repair ofosteochondral defects. Mobilizedpluripotent MSCs from the subchondralbone migrate into the defect filled withthe clot, differentiate into chondrocytesand osteoblasts, and form a repairtissue over time. The additionalapplication of a bone marrow aspirate(BMA) to the procedure of marrow

stimulation is thought to enhancecartilage repair, as marrow itself is botha source of MSCs, providing a cellpopulation capable of chondrogenesisand of various growth factorsstimulating cartilage repair. Moreover,the BMA clot provides a threedimensional environment, possiblyfurther supporting chondrogenesis andprotecting the subchondral bone fromstructural alterations.

Why use BMA as a source of stemcells?

Embryonic stem cells areconsidered the holy grail of stem cells,as they are totipotent and have thepotential to differentiate into any celllineage. But their use is associated withsignificant ethical issues andconsiderations, as they require harvestfrom embryonic tissue.

Adult stem cells are multipotentand can develop into cells that supportthe tissue of origin. They can bestimulated to irreversibly change theircell lineage in a process termed astransdifferentiation, where cellstransform into a different cell type.

MSCs are found not only inbone marrow but also in many othermesenchymal tissues, including


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adipose tissue, synovium and blood.However, MSCs from these differentmesenchymal tissues differ in theirdifferentiation potential, with thosederived from bone marrow being themost multipotent. Also, bone marrow-derived MSCs are innate, they havethe appropriate host majorhistocompatibility complex (MHC)allowing them to avoid destruction bythe immune system. Bone marrowaspirate (BMA) is relative easy toharvest, and is a less controversialsource of MSCs with the requiredproperties for use in regenerativeorthopaedics.

Need for concentration of BMA:The main concern in using BMA tostimulate tissue repair / regeneration

is that only 0.001% - 0.01% ofnucleated cells within BMA are MSCs.To address this issue, various protocolshave been developed to concentratethe nucleated cell numbers to producebone marrow aspirate concentrate(BMAc). Sufficient amount of MSCsare needed to provide an effectiveenvironment of healing andregeneration.

Indications for use of BMAcinclude bone defects / non-unions, AVNparticularly of the femoral head,cartilage / osteochondral defects of theknee, talus, etc and tendon injuriessuch as rotator cuff tears, Achillestendinopathy. It is a very important first-line treatment option for smallsymptomatic articular cartilage defects.

Preparation of BMAC:

I. Aspirate 35 ml bone marrow in a syringe pre-filled with ACD-A. This can

85

Technique used for cartilage /osteochondral defects: The basicprinciple is to establish acommunication of the cartilage defectwith the subchondral bone marrowcompartment. The cartilage defect issurgically prepared by removal ofcartilage fragments and generation ofstable and vertically oriented marginsof the peripheral cartilage. The next

step involves preparation of the bonydefect base. The entire layer ofcalcified cartilage has to be removed,thereby exposing the superficial partof the subchondral bone plate withoutdamaging it.

This is followed by marrowstimulation, performed bymicrofracture, subchondral drilling orabrasion arthroplasty.

be achieved by percutaneous aspiration under local / general anaesthesia fromthe iliac crest. Trocar placement can be done under fluoroscopy to allow formaximal depth of harvest.

II. 1st centrifugation at 3500 rpm for 6 minutes.

III. Rotate marrow chamber to transfer all buffy coat to plasma chamber.

IV: 2nd centrifugation after inverting the chamber, at 3300 rpm for 5 minutes.

V. Harvest BMAC for use.


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After marrow stimulation, the bonemarrow containing mesenchymal stemcells ascends from the marrow cavityof the underlying subchondral bone viathe channels generated by the marrowstimulation procedures. These defectsare filled with a clot of autologousBMAC, containing mesenchymal stem

cells and growth factors, which favornew tissue formation. Defects thuscontain bone marrow both from thesubchondral bone and the additionalBMAC application, and gradually a car-tilaginous repair tissue forms withinthem.

Trochlea lesion in right knee

(A) Lesion after debridement and drilling. The holes are 7 mm deep and placed 2 to 3mm apart. (B) The same lesion is injected with BMAC under arthroscopic guidance.

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Advantages: It is safe asautologous blood is used, thusassociated with a lower risk of diseasetransmission or rejection reaction, andcost-effective. It is also easy to use,effective, and being a single-stageprocedure, has better patientsatisfaction. Its use may help inavoiding major surgery in the future forOA knees.

Results with BMAC: In 2002,Wakitani et al. conducted a study with2 groups of 12 patients each, with OAknee undergoing high tibialosteotomies. One group receivedBMAC during surgery and the otherserved as a control. They found asignificant arthroscopic and histologicalimprovement in the BMAC group, butno clinical advantage.

In 2008, Centeno et al. showedthat intra-articular injection of BMACinto a knee with symptomatic andradiographic degenerative jointdisease resulted in significant cartilagegrowth, decreased pain and increasedjoint mobility.

In 2012, Emadedin et al. reportedsatisfactory improvement in pain andfunctional status after intra-articular

injection of BMAC in 6 patients withknee OA. They performed an MRI ofthese patients at 6 months follow up,which demonstrated an increase incartilage thickness, extension of therepair tissue over the subchondralbone and a considerable decrease inthe size of edematous subchondralpatches.

In 2013, Orozco et al. conductedanother clinico-radiological pilot studyand treated 12 patients with OA kneewith intra-articular BMAC injection, andevaluated their clinical and radiological(MRI) outcome at 1 year follow up.There was significant improvementboth clinically - improvement in pain,disability and quality of life, and,radiologically - highly significantdecrease of poor cartilage areas alongwith improvement of cartilage qualityin 11 of the 12 patients.

In 2014, Kim et al. evaluated theclinical efficacy of intra-articularinjection of BMAC with adipose tissuein 41 patients with OA knee. There wasimprovement in both the pain VASscore and the functional scores (IKDC,SF-36, Lysholm Knee Questionnaire)at 12 months follow-up.


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In 2016, Chahla et al. performeda systematic review of 11 studies onthe outcomes of bone marrow aspirateconcentrate for the treatment ofchondral defects and osteoarthritis ofthe knee. Of these, 5 were prospectivestudies, 1 was a retrospective study, 2were case series, and 3 were casereports. They reported good toexcellent overall outcomes with the useof BMAC for the treatment of earlyknee osteoarthritis and moderate focalchondral defects.

In 2017, Moatshe et al. conductedanother systematic review of studieson the efficacy of the biologictreatments utilized in knee pathologies,including PRP and BMAC. They found

that, although most of these studiesshowed good clinical and functionaloutcomes with the use of thesetechniques, with an acceptable safetyprofile, they lack enough power andfollow-up time for the evidence to becompelling.

In 2018, Themistocleous et al.performed a study of 233 patients withidiopathic OA of the knee, treated witha single intra-articular BMAC injection.At a mean follow-up period of 11months, they reported that the patientsshowed significant clinical andfunctional improvement with respect tothe Numeric Pain Scale and the OxfordKnee Score. They found nocomplication in any patient.

Conclusion

To summarize, many important advancements have been made inOrthobiologics and these therapies are still evolving . We need a clearunderstanding of the basic underlying pathology of OA and the mechanismby which these Orthobiologics can be helpful . Osteoarthritis is a multifaceteddisease involving not only the hyaline cartilage but also fibrous capsule ,meniscus and the subchondral bone as the other variables of OA knee suchas limb malalignment, ligamentous insufficiency and muscular imbalanceshave to be considered . Most of the studies suggest good outcomes withusing Orthobiologics (PRP, BMAc) but they lack enough power and followup. We need clear understanding of the graft choice and patient selection inthe treatment of early OA. In order to achieve this, we suggest the need forconducting further well designed randomized control trails with definiteprotocols to elucidate the real efficacy of these therapies.

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BMP’s in Orthopaedics

1. Urist MR, Mikulski AJ, Nakagawa M,Yen K (1977) A bone matrix calcification-initiator noncollagenous protein. Am JPhysiol 232(3): C115–C127

2. Bishop GB, Einhorn TA (2007) Currentand future clinical applications of bonemorphogenetic proteins in orthopaedictrauma surgery. Int Orthop 31(6):721–727.doi:10.1007/s00264-007-0424-8

3. Lissenberg-Thunnissen SN, de GorterDJ, Sier CF, Schipper IB (2011) Use andefficacy of bone morphogenetic proteinsin fracture healing. Int Orthop 35(9):1271–1280. doi:10.1007/s00264-011- 1301-z

4. Vukicevic S, Oppermann H, VerbanacD, Jankolija M, Popek I, Curak J, BrkljacicJ, Pauk M, Erjavec I, Francetic I, Dumic-Cule I, Jelic M, Durdevic D, Vlahovic T,Novak R, Kufner V, Bordukalo Niksic T,Kozlovic M, Banic Tomisic Z, Bubic-SpoljarJ, Bastalic I, Vikic-Topic S, Peric M, PecinaM, Grgurevic L (2014) The clinical use ofbone morphogenetic proteins revisited: anovel biocompatible carrier deviceOSTEOGROW for bone healing. IntOrthop 38(3): 635–647. doi:10.1007/s00264-013-2201-1

5. Fu R, Selph S, McDonagh M, PetersonK, Tiwari A, Chou R, Helfand M (2013)

Bibliography

Effectiveness and harms of recombinanthuman bone morphogenetic protein-2 inspine fusion: a systematic review andmeta-analysis. Ann Intern Med 158(12):890-902. doi:10.7326/ 0003-4819-158-12-201306180-00006

6. Simmonds MC, Brown JV, Heirs MK,Higgins JP, Mannion RJ, Rodgers MA,Stewart LA (2013) Safety andeffectiveness of recombinant human bonemorphogenetic protein-2 for spinal fusion:a meta-analysis of individual-participantdata. Ann Intern Med 158 (12):877-889.d o i : 1 0 . 7 3 2 6 / 0 0 0 3 - 4 8 1 9 - 1 5 8 - 1 2 -201306180-00005

7. Delimar D, Smoljanovic T, Bojanic I(2012) Could the use of bonemorphogenetic proteins in fracture healingdo more harm than good to our patients?Int Orthop 36 (3):683; author reply 685.doi:10.1007/ s00264-011-1397-1

8. Bong MR, Capla EL, Egol KA, SorkinAT, Distefano M, Buckle R, Chandler RW,Koval KJ (2005) Osteogenic protein-1(bone morphogenic protein-7) combinedwith various adjuncts in the treatment ofhumeral diaphyseal nonunions. Bull HospJt Dis 63(1–2): 20–23

9. Dimitriou R, Dahabreh Z, Katsoulis E,Matthews SJ, Branfoot T, Giannoudis PV

90 Orthobiologics

(2005) Application of recombinant BMP-7on persistent upper and lower limb non-unions. Injury 36 Suppl 4:S51-59.doi:10.1016/j.injury.2005.10.010

10. Moghaddam A, Elleser C, Biglari B,Wentzensen A, Zimmermann G (2010)Clinical application of BMP 7 in long bonenon-unions. Arch Orthop Trauma Surg130(1):71–76. doi:10.1007/s00402-009-0982-x

11. Ronga M, Baldo F, Zappala G,Cherubino P (2006) Recombinant humanbone morphogenetic protein-7 fortreatment of long bone non- union: anobservational, retrospective, non-randomized study of 105 patients. Injury37 Suppl 3:S51-56. doi:10.1016/j.injury.2006.08.024

12. Evans RO, Goldberg JA, Bruce WJ,Walsh W (2004) Reoperated clavicularnonunion treated with osteogenic protein1 and electrical stimulation. J ShoulderElbow Surg 13 (5):573-575. doi:10.1016/S105827460400076X

13. Clark RR, McKinley TO (2010)Bilateral olecranon epiphyseal fracturenon-union in a competitive athlete. IowaOrthop J 30: 179–181

14. Friedlaender GE, Perry CR, Cole JD,Cook SD, Cierny G, Muschler GF, ZychGA, Calhoun JH, LaForte AJ, Yin S (2001)Osteogenic protein-1 (bonemorphogenetic protein-7) in the treatment

of tibial nonunions. J Bone Joint Surg Am83-A Suppl 1(Pt 2):S151–S158

15. Zimmermann G, Wagner C,Schmeckenbecher K, Wentzensen A,Moghaddam A (2009) Treatment of tibialshaft non-unions: bone morphogeneticproteins versus autologous bone graft.Injury 40 Suppl 3:S50-53. doi:10.1016/S0020-1383(09)70012-9

16. Kanakaris NK, Calori GM, Verdonk R,Burssens P, De Biase P, Capanna R,Vangosa LB, Cherubino P, Baldo F,Ristiniemi J, Kontakis G, Giannoudis PV(2008) Application of BMP-7 to tibial non-unions: a 3-year multicenter experience.Injury 39 Suppl 2:S83- 90. doi:10.1016/S0020-1383(08)70019-6

17. Obert L, Deschaseaux F, Garbuio P(2005) Critical analysis and efficacy ofBMPs in long bones non-union. Injury 36Suppl 3:S38- 42. doi:10.1016/j.injury.2005.07.033

18. Wysocki RW, Cohen MS (2007)Ectopic ossification of the triceps muscleafter application of bone morphogeneticprotein-7 to the distal humerus forrecalcitrant nonunion: a case report. JHand Surg Am 32 (5):647-650.doi:10.1016/j.jhsa.2007.03.001

19. Boraiah S, Paul O, Hawkes D,Wickham M, Lorich DG (2009)Complications of recombinant humanBMP-2 for treating complex tibial plateau

91

fractures: a preliminary report. Clin OrthopRelat Res 467(12):3257–3262.doi:10.1007/s11999-009-1039-8

20. Bilic R, Simic P, Jelic M, Stern-Padovan R, Dodig D, van MeerdervoortHP, Martinovic S, Ivankovic D, Pecina M,Vukicevic S (2006) Osteogenic protein-1(BMP-7) accelerates healing of scaph- oidnon-union with proximal pole sclerosis. IntOrthop 30(2):128– 134. doi:10.1007/s00264-005-0045-z

21. Bibbo C, Patel DV, Haskell MD (2009)Recombinant bone morphogeneticprotein-2 (rhBMP-2) in high-risk ankle andhindfoot fusions. Foot Ankle Int 30 (7):597-603. doi:10.3113/FAI.2009.0597

22. Schuberth JM, DiDomenico LA,Mendicino RW (2009) The utility andeffectiveness of bone morphogeneticprotein in foot and ankle surgery. J FootAnkle Surg 48 (3):309-314. doi:10.1053/j.jfas.2009. 01.011

23. El-Amin SF, Hogan MV, Allen AA,Hinds J, Laurencin CT (2010) Theindications and use of bonemorphogenetic proteins in foot, ankle, andtibia surgery. Foot Ankle Clin 15 (4):543-551. doi:10. 1016/j.fcl.2010.08.001

24. Fourman MS, Borst EW, Bogner E,Rozbruch SR, Fragomen AT (2014)Recombinant Human BMP-2 Increasesthe Incidence and Rate of Healing inComplex Ankle Arthrodesis. Clin Orthop

Relat Res 472(2):732–739. doi:10.1007/s11999-013-3261-7

25. Hawkins BJ (2010) Biologics in footand ankle surgery. Foot Ankle Clin 15(4):577-596. doi:10.1016/j.fcl.2010.07.003

26. DeVries JG, Nguyen M, Berlet GC,Hyer CF (2012) The effect of recombinantbone morphogenetic protein-2 in revisiontibiotalocalcaneal arthrodesis: utilization ofthe Retrograde Arthrodesis IntramedullaryNail database. J Foot Ankle Surg 51(4):426-432. doi:10.1053/j.jfas.2012.03.007

27. Kanakaris NK, Mallina R, Calori GM,Kontakis G, Giannoudis PV (2009) Use ofbone morphogenetic proteins inarthrodesis: clinical results. Injury 40Suppl 3:S62-66. doi:10.1016/S0020-1383(09) 70014-2S0020-1383 (09)70014-2

28. Pecina M, Jelic M, Martinovic S, HasplM, Vukicevic S (2002) Articular cartilagerepair: the role of bone morphogeneticproteins. Int Orthop 26(3):131–136.doi:10.1007/s00264-002-0338-4

29. Tannoury C, An HS (2014)Complications with Use of BoneMorphogenetic Protein-2 (BMP-2) in SpineSurgery. Spine J. doi: 10.1016/j.spinee.2013.08.060

30. Singh K, Ahmadinia K, Park DK,Nandyala SV, Marquez-Lara A, Patel AA,

92 Orthobiologics

Fineberg SJ (2014) Complications ofspinal fusion with utilization of bonemorphogenetic protein: a systematicreview of the literature. Spine (Phila Pa1976) 39(1):91–101. doi:10.1097/BRS.0000000000000004

31. Vaidya R, Sethi A, Bartol S, JacobsonM, Coe C, Craig JG (2008) Complicationsin the use of rhBMP-2 in PEEK cages forinterbody spinal fusions. J Spinal DisordTech 21 (8):557-562. doi:10.1097/BSD.0b013e31815ea897

32. Balseiro S, Nottmeier EW (2010)Vertebral osteolysis originating fromsubchondral cyst end plate defects intransforaminal lumbar interbody fusionusing rhBMP-2. Report of two cases.Spine J 10 (7): e6-e10. doi:10.1016/j.spinee.2010.04.013

33. Helgeson MD, Lehman RA, Jr.,Patzkowski JC, Dmitriev AE, Rosner MK,Mack AW (2011) Adjacent vertebral bodyosteolysis with bone morphogeneticprotein use in transforaminal lumbarinterbody fusion. Spine J 11 (6):507-510.doi:10.1016/j.spinee. 2011.01.017

34. Saigal G, Quencer R, Guest JD,Cristescu MM, Lebwohl N (2012) Vertebralbody osteolysis following the use of bonemorphogenetic protein in spinal surgery:a mimicker of infection. J Neuroradiol 39(5):354-359. doi:10.1016/j.neurad.2012.03.005

35. Smoljanovic T, Bojanic I, Cimic M(2009) Bone morphogenetic protein. JNeurosurg Spine 11 (1):92-93; authorreply 93-94. doi: 10.3171/2009.2.SPINE08953L

36. Deutsch H (2010) High-dose bonemorphogenetic protein-induced ectopicabdomen bone growth. Spine J 10 (2):e1-4. doi:10.1016/j. spinee.2009.10.016

37. Anderson CL, Whitaker MC (2012)Heterotopic ossification associated withrecombinant human bone morphogeneticprotein-2 (infuse) in posterolateral lumbarspine fusion: a case report. Spine (PhilaPa 1976) 37(8):E502–E506. doi:10.1097/BRS.0b013e318238870b

38. Joseph V, Rampersaud YR (2007)Heterotopic bone formation with the useof rhBMP2 in posterior minimal accessinterbody fusion: a CT analysis. Spine(Phila Pa 1976) 32 (25):2885-2890.doi:10.1097/BRS.0b013e31815b7596

39. Chen NF, Smith ZA, Stiner E, ArminS, Sheikh H, Khoo LT (2010) Symptomaticectopic bone formation after off-label useof recombinant human bonemorphogenetic protein-2 in transforaminallumbar interbody fusion. J NeurosurgSpine 12(1):40–46. doi:10.3171/2009.4.SPINE0876

40. Glassman SD, Gum JL, Crawford CH,3rd, Shields CB, Carreon LY (2011)Complications with recombinant human

93

bone morphogenetic protein-2 inposterolateral spine fusion associated witha dural tear. Spine J 11 (6):522-526.doi:10.1016/j.spinee.2010.05.016

41. Choudhry OJ, Christiano LD, SinghR, Golden BM, Liu JK (2012) Bonemorphogenetic protein-inducedinflammatory cyst formation after lumbarfusion causing nerve root compression. JNeurosurg Spine 16(3):296–301.doi:10.3171/2011.11.SPINE11629

42. Smucker JD, Rhee JM, Singh K, YoonST, Heller JG (2006) Increased swellingcomplications associated with off-labelusage of rhBMP-2 in the anterior cervicalspine. Spine (Phila Pa 1976) 31 (24):2813-2819. doi:10.1097/01.brs.0000245863.52371.c2

43. Perri B, Cooper M, Lauryssen C,Anand N (2007) Adverse swellingassociated with use of rh-BMP-2 inanterior cervical discectomy and fusion:a case study. Spine J 7 (2):235-239.doi:10.1016/j.spinee. 2006.04.010

44. Shahlaie K, Kim KD (2008)Occipitocervical fusion using recombi-nant human bone morphogenetic protein-2: adverse effects due to tissue swellingand seroma. Spine (Phila Pa 1976) 33(21):2361- 2366. doi:10.1097/BRS.0b013e318183971d

45. Anticevic D, Jelic M, Vukicevic S(2006) Treatment of a congenital

pseudarthrosis of the tibia by osteogenicprotein-1 (bone morphogenetic protein-7): a case report. J Pediatr Orthop B 15(3):220-221. doi: 10.1097/01.bpb.0000194439.75378.ac

46. Spiro AS, Babin K, Lipovac S, StengerP, Mladenov K, Rupprecht M, Rueger JM,Stuecker R (2011) Combined treatment ofcongenital pseudarthrosis of the tibia,including recombinant human bone mor-phogenetic protein-2: a case series. JBone Joint Surg Br 93 (5):695- 699.doi:10.1302/0301-620X.93B5.25938

47. Pannier S (2011) Congenitalpseudarthrosis of the tibia. OrthopTraumatol Surg Res 97 (7):750-761.doi:10.1016/j.otsr.2011.09.001 48.Richards BS, Oetgen ME, Johnston CE(2010) The use of rhBMP-2 for thetreatment of congenital pseudarthrosis ofthe tibia: a case series. J Bone Joint SurgAm 92 (1):177-185. doi:10.2106/JBJS.H.01667

49. Cheng TL, Murphy CM, Cantrill LC,Mikulec K, Carpenter C, Schindeler A,Little DG (2014) Local delivery ofrecombinant human bone morphogeneticproteins and bisphosphonate via sucroseacetate isobutyrate can prevent femoralhead collapse in Legg-Calve-Perthesdisease: a pilot study in pigs. Int Orthop38(7):1527–1533. doi:10. 1007/s00264-013-2255-0

94 Orthobiologics

50. Steib JP, Bouchaib J, Walter A,Schuller S, Charles YP (2010) Could anosteoinductor result in degeneration of aneurofibroma in NF1? Eur Spine J19(Suppl 2):S220–S225. doi:10.1007/s00586-010-1416-8

Platelet biology and introductionto Platelet Rich Plasma

1.Malanga GA, Goldin M. PRP: review ofthe current evidence for musculoskeletalconditions. CurrPhys Med Rehabil Rep2014;2:1–5.

2. Davis VL, Abukabda AB, Radio NM, etal. Platelet-rich preparations to improvehealing. Part I: workable options for everysize practice. J Oral Implantol 2014;40(4):500–10.

3. Boswell SG, Cole BJ, Sundman EA, etal. Platelet-rich plasma: a milieu ofbioactive factors. Arthroscopy2012;28(3):429–39.

4. Blair P, Flaumenhaft R. Platelet alpha-granules: basic biology and clinicalcorrelates. Blood Rev 2009;23(4):177–89.

5. Hoemann CD, Chen G, Marchand C, etal. Scaffold-guided subchondral bonerepair: implication of neutrophils andalternatively activated arginase-11macrophages. Am J Sports Med2010;38(9):1845–56.

6. 14. Assirelli E, Filardo G, Mariani E, etal. Effect of two different preparations of

platelet-rich plasma on synoviocytes.Knee Surg Sports TraumatolArthrosc2015;23(9):2690–703.

7. Braun HJ, Kim HJ, Chu CR, et al. Theeffect of platelet-rich plasma formulationsand blood products on humansynoviocytes: implications for intra-articular injury and therapy. Am J SportsMed 2014;42(5):1204–10.

8.Javier Ramos-Torrecillas,Elvira DeLuna-Bertos,OlgaGarcía-Martínez,Concepción Ruiz. Clinical Utility of GrowthFactors and Platelet-Rich Plasma inTissue Regeneration: A Review.WOUNDS. 2014;26(7):207-213.

9. Peter I-Kung Wu, Robert Diaz,JoanneBorg-Stein. Platelet-Rich Plasma, PhysMed RehabilClin N Am 27 (2016) 825–853.

10. Platelet Rich Plasma (PRP)Guidelines, © International CellularMedicine Society – 2011. Available atwww.cellmedicinesociety.org.

11. Shetty VD, Dhillon M, Hegde C, JagtapP, Shetty S. A study to compare theefficacy of corticosteroid therapy withplatelet-rich plasma therapy in recalcitrantplantar fasciitis: a preliminary report. FootAnkle Surg. 2014; 20(1):10-13.

12. Kumar V, Millar T, Murphy PN, CloughT. The treatment of intractable planterfasciitis with platelet-rich plasma injection.

95

Foot (Edinb). 2013; 23:74-77.

14.F. SAY, E. TÜRKELI, M. BÜLBÜL. IsPlatelet-Rich Plasma Injection an EffectiveChoice in Cases of Non-Union? .Actachirurgiae orthopaedicae ettraumatologiae Cechoslovaca2014.81(5):340-345 .

15. Mishra A, Pavelko T (2006) Treatmentof chronic elbow tendinosis with bufferedplatelet-rich plasma. Am J Sports Med;34:1774–1778.

16. Sebastian Lippross, Mauro Alini.Platelet-rich plasma for bone healing— touse or not to use?Avaialable at https://p d f s . s e m a n t i c s c h o l a r. o r g / 6 2 e 7 /d0bcf3f92248aa4f0606b0cba8f3de695536.pdf.

17.Dhillon MS, Behera P, Patel S, ShettyV. Orthobiologics and platelet rich plasma.Indian J Orthop 2014;48:1-9.

Platelet Rich Plasma extraction withlow cost assembled kit.

1.Eduardo Anitua, Roberto Prado, MikelSánchez, Gorka Orive. Platelet-RichPlasma: Preparation and Formulation.Oper Tech Orthop ; 2012:22:25-32.

2. Alsousou.j, Thompson.M, Hulley.p,Noble.A, Willett.K. The biology of platelet-rich plasma and its application in traumaand orthopaedic surgery. J Bone JointSurg [Br]2009;91-B:987-96.

3.Marx RE. Platelet-rich plasma (PRP):what is PRP and what is not PRP? ImplantDent. 2001;10:225-8.

4.Augustus D. Mazzocca, Mary Beth R.McCarthy, David M. Chowaniec, Mark P.Cote, Anthony A. Romeo, James P.Bradley etal. Platelet-Rich Plasma DiffersAccording to Preparation Method andHuman Variability. Bone Joint Surg Am.2012;94:308-16.

5.Platelet Rich Plasma (PRP) guidelines,international celluar medicinesociety.2011[ last accessed 2016 sept 10].Available from:http://www.cellmedicinesociety.org.

6.Paola Romina Amable,Rosana BizonVieira Carias,Marcus Vinicius TellesTeixeira,Ítalo da Cruz Pacheco,RonaldoJosé Farias Corrêa do Amaral,José MauroGranjeiro et al. Platelet-rich plasmapreparation for regenerative medicine:optimization and quantification ofcytokines and growth factors. Stem CellResearch & Therapy2013;4:67-78.

7. Han-tung Liao, Kacey Marra,J PeterRubin.Application of platelet –rich plasmaand platelet rich fibrin in fat grafting:basicscience and literature review.tissueenginering2014;20(4):267-72.

8.Amanda G. M. Perez, José Fábio S. D.Lana, Ana Amélia Rodrigues, AngelaCristina M. Luzo, William D. Belangero,

96 Orthobiologics

Maria Helena A. Santana. RelevantAspects of Centrifugation Step in thePreparation of Platelet-Rich Plasma. ISRNHematology 2014;1-8.

9.Olivier Bausset, Laurent Giraudo, JulieVeran, Jeremy Magalon, Jean-MarieCoudreuse, Guy Magalon etal .Formulation and Storage of Platelet-RichPlasma Homemade Product.BioResearch Open Access 2012;1:115-21.

10.Yavuz kececi, sibel ozsu,Oktay bilgar.Acost effectivr method for obtainingstandard platelet richplasma.wounds2014;28(8):232-8.

11.S.J Slichter. Harker. Preparation andstorage of platelet concentrates:1. Factorsinfluencing the harves of viable plateletsfrom whole blood.Br j Haematol1976;34(3):395-402.

12.Jennifer E. Woodell-May, Dayna N.Ridderman, Matthew J. Swift,y JoelHiggins. Producing Accurate PlateletCounts for Platelet Rich Plasma:Validation of a Hematology Analyzer andPreparation Techniques for Counting. Thejournal of craniofacial surgery 2005;16(5):749-56.

13.Schneider bs, Tiidus pp. neutrophilinfilitration in exercise injured skeletalmuscle. Sportsmed 2007;37:837-56.

14.Michael p Hall, PhillipA Band, Robert

J Meislin, Laith M Jazrawi,Dennis ACardone. Platelet rich plasma:currentconcepts and application in sportsmedicine. Jr AAOS2009;17(10):602-8.

15.Mototsugu Fukaya, Asako Ito. A neweconomic method for preparing platelet –rich plasma . j PRSGO.2014;1-7.

16. Masoomeh Gholami, Hamid Ravaghi,Masoud Salehi, Amirhosein Abedi Yekta,Shila Doaee, Ebrahim Jaafaripooyan. Asystematic review and meta-analysis ofthe application of platelet rich plasma insports medicine. Electronic Physician2016; 8:2325-32.

Platelet-rich plasma therapy intendinopathies

1. Scher D, Jr Belmont P, Owens B, . Theepidemiology of plantar fasciitis, LER, April2010; www.lermagazine.com

2. Alves R. Grimalt R. A Review of Platelet-Rich Plasma: History, Biology, Mechanismof Action, and Classification SkinAppendage Disord 2018;4:18–24

3.Magalon J, Bausset O, Serratrice N, etal. Characterization and comparison of 5platelet-rich plasma preparations in asingle-donor model. Arthroscopy. 2014;30: 629–638

4.Blajchman M. Novel platelet products,substitutes and alternatives. Transfus ClinBiol. 2001; 8:267–271

97

5.Dragoo J, Braun H, Durham J, RidleyB, Odegaard J, Luong R, Arnoczky S.Comparison of the acute inflammatoryresponse of two commercial platelet-richplasma systems in healthy rabbit tendons.Am J Sports Med. 2012;40:1274–1281

6. Adrian D, Lawrence E, Malcolm R, andJason L. Dragoo J. Current ClinicalRecommendations for the use of Platelet-Rich Plasma. Curr Rev MusculoskeletMed. 2018 Dec; 11(4): 624–634

7. Arirachakaran A, Sukthuayat A,Sisayanarane T, Laoratanavoraphong S,Kanchanatawan W, Kongtharvonskul J.Platelet-rich plasma versus autologousblood versus steroid injection in lateralepicondylitis: a systematic review andnetwork meta-analysis. J OrthopTraumatol. 2016;17:101–112

8. Krogh T, Bartels E, Ellingsen T,Stengaard-Pedersen K, Buchbinder R,Fredberg U, Bliddal H, Christensen R.Comparative effectiveness of injectiontherapies in lateral epicondylitis: asystematic review and network meta-analysis of randomized controlled trials.Am J Sports Med. 2013;41:1435–1446

9. Dragoo J, Wasterlain A, Braun H, NeadK. Platelet-rich plasma as a treatment forpatellar tendinopathy: a double-blind,randomized controlled trial. Am J SportsMed. 2014;42:610–618

10. Shetty V, Dhillon M, Hegde C, JagtapP, Shetty S. A study to compare theefficacy of corticosteroid therapy withplatelet-rich plasma therapy in recalcitrantplantar fasciitis: a preliminary report. FootAnkle Surg. 2014;20:10–13

11. Mahindra P, Yamin M, Selhi H, SinglaS, Soni A. Chronic plantar fasciitis: effectof platelet-rich plasma, corticosteroid, andplacebo. Orthopedics. 2016;39:e285–e289

12. Monto R. Platelet-rich plasma efficacyversus corticosteroid injection treatmentfor chronic severe plantar fasciitis. FootAnkle Int. 2014;35:313–318

13. Yang W, Han Y, Cao X, Pan J, Zeng L,Lin J, et al. Platelet-rich plasma as atreatment for plantar fasciitis: a meta-analysis of randomized controlled trials.Medicine (Baltimore) 2017;96:e8475

14. Singh P, Madanipour S, Bhamra J, GillI. A systematic review and meta-analysisof platelet-rich plasma versuscorticosteroid injections for plantarfasciopathy. Int Orthop. 2017;41:1169–1181

15. de Vos RJ, Weir A, Tol J, Verhaar J,Weinans H, van Schie H. No effects ofPRP on ultrasonographic tendon structureand neovascularisation in chronicmidportion Achilles tendinopathy. Br JSports Med. 2011;45:387–392

98 Orthobiologics

PRP in OA Knee:Consensus Document

1. Buckwalter JA, Mankin HJ (1997)Articular cartilage: degeneration andosteoarthrosis, repair, regeneration, andtransplantation. J Bone Joint Surg Am79A(4):612–632.

2. Gobbi A (2010) New possibilities forcartilage repair. Eur Musculoskelet Rev5(2):64–68

3. Tran-Khanh N, Hoemann CD, McKeeMD, Henderson JE, Buschmann MD(2005) Aged bovine chondrocytesdisplay a diminished capacity to producea collagen-rich, mechanically functionalcartilage extracellular matrix. J Orthop Res23:1354–1362

4. Pietrzak WS, Eppley BL (2007) Plateletrich plasma: biology and new technology.J Craniofac Surg 16(6):1043–1054

5. Frisbie DD, Kawcak CE, Werpy NM,Park RD, McIlwraith CW (2007) Clinical,biochemical, and histologic effects of intra-articular administration of autologousconditioned serum in horses withexperimentally induced osteoarthritis.Am J Vet Res 68:290–296

6. Treatment With Platelet-Rich PlasmaIs More Effective Than Placebo for KneeOsteoarthritis A Prospective, Double-Blind, Randomized Trial Sandeep Patel ,Mandeep S. Dhillon, Sameer Aggarwal,Neelam Marwaha, Ashish Jain, MD. The

American Journal of Sports Medicine, Vol.XX, No. X DOI: 10.1177/0363546512471299 2013

Bone Marrow Aspirate Concentrate

1. Arianna L Gianakos, Li Sun, JayN Patel,Donald M Adams,Frank A Liporace.Clinical application of concentrated bonemarrow aspirate in orthopaedics:Asystemic review. World J Orthop2017.8(6):491-506.

2. Jorge Chahla,Sandeep M, Mark Ecinque et al.bone marrow aspirateconcentrate harvesting and processingtechnique. Arthroscopy Techniques2017.6(2):441-5.

3. Mohammed A. Imam, Samer SSmahumoud et al. A systematic review ofthe concept and clinical applications ofbone marrow Aspirate Concentrate inOrthopaedics. SICOT j2017.3:17.

4. Centeno C, pitts J, Al-sayeghH,Freeman M. Efficacy of autologous bonemarrow concentrate for knee osteoarthritiswith and without adipose graft. BiomedRes Int.2014:370621.

5. Hauser RA, Orlofsky A. Regenerativeinjection therapy with whole bone marrowaspirate for degenerative joint diseases :a case series. Clin Med Insights ArthritisMusculoskeletal disord. 2013;6:65-72.

6. Kim JD,LeeGW,Jung GH et al. clinicaloutcome of autologous bone marrow

99

aspirate concentrate(BMAC) injection indegenerative arthritis of the knee. Eur JOrthopSurg Traumatol.2014;24:1505-11.

7. Hernigou P, Mathieu G, Poignard A,Manicom O, Manicom O, Beaujean F,Beaujean F, Rouard H (2006)Percutaneous autologous bone-marrowgrafting for nonunions. Surgical technique.J Bone Joint Surg Am 88, 322–327

8. Hernigou P, Beaujean F (2002)Treatment of osteonecrosis withautologous bone marrow grafting.ClinOrthopRelat Res 405, 14–23.

9.Giannini S, Buda R, Cavallo M, Ruflli A,Cenacchi A, Cavallo C, Vannini F (2010)Cartilage repair evolution in posttraumaticosteochondral lesions of the talus: fromopen eld autologous chondrocyte to bone-marrow-derived cells transplantation.Injury 41(11), 1196–1203.

10.Hernigou P, FlouzatLachaniette CH,Delambre J, Zilber S, Dufet P, ChevallierN, Rouard H (2014) Biologic augmentationof rotator cuff repair with mesenchymalstem cells during arthroscopy improveshealing and prevents further tears: acasecontrolled study. IntOrthop 38(9),1811–1818.

BMAC in Non Union of Fractures

1. Bastos Filho R, Lermontov S,Borojevic R, Schott PC, Gameiro VS,Granjeiro JM. Cell therapy ofpseudarthrosis. Acta Ortop Bras 2012;

20: 270-273 [PMID: 24453616 DOI:10.1590/S1413-7852 2012000500005]

2. Desai P, Hasan SM, Zambrana L,Hegde V, Saleh A, Cohn MR, Lane JM.Bone Mesenchymal Stem Cells withGrowth Factors Successfully TreatNonunions and Delayed Unions. HSS J2015; 11: 104-111 [PMID: 26140028 DOI:10.1007/s11420-015-9432-1]

3. Garnavos C, Mouzopoulos G, MorakisE. Fixed intramedullary nailing andpercutaneous autologous concentratedbone-marrow grafting can promote bonehealing in humeral-shaft fractures withdelayed union. Injury 2010; 41: 563-567[PMID: 19740464 DOI: 10.1016/j.injury.2009.08.003]

4. Guimarães JA, Duarte ME, FernandesMB, Vianna VF, Rocha TH, Bonfim DC,Casado PL, do Val Guimarães IC, VelardeLG, Dutra HS, Giannoudis PV. The effectof autologous concentrated bonemarrowgrafting on the healing of femoral shaftnon-unions after locked intramedullarynailing. Injury 2014; 45 Suppl 5: S7-S13[PMID: 25528626 DOI: 10.1016/S0020-1383(14)70013-0]

5. Hernigou P, Guissou I, Homma Y,Poignard A, Chevallier N, Rouard H,Flouzat Lachaniette CH. Percutaneousinjection of bone marrow mesenchymalstem cells for ankle non-unions decreasescomplications in patients with diabetes. Int

100 Orthobiologics

Orthop 2015; 39: 1639-1643 [PMID:25795249 DOI: 10.1007/s00264-015-2738-2]

6. Hernigou P, Poignard A, Beaujean F,Rouard H. Percutaneous autologousbone-marrow grafting for nonunions.Influence of the number and concentrationof progenitor cells. J Bone Joint Surg Am2005; 87: 1430-1437 [PMID: 15995108DOI:10.2106/JBJS.D.02215]

7. Ismail HD, Phedy P, Kholinne E, DjajaYP, Kusnadi Y, Merlina M, Yulisa ND.Mesenchymal stem cell implantation inatrophic non-union of the long bones: Atranslational study. Bone Joint Res 2016;5: 287-293 [PMID: 27412657 DOI:10.1302/2046-3758.57.2000587]

8. Le Nail LR, Stanovici J, Fournier J,Splingard M, Domenech J, Rosset P.Percutaneous grafting with bone marrowautologous concentrate for open tibiafractures: analysis of forty three cases andliterature review. Int Orthop 2014; 38:1845-1853 [PMID: 24728310 DOI:10.1007/ s00264-014-2342-x]

Mesenchymal Stem Cells inManagement of Avascular NecrosisFemur Head

1. Sen RK. Management of avascularnecrosis of femoral head at pre-collapsestage. Indian J Orthop. 2009;43(1):6-16.2. Tripathy SK, Goyal T, Sen RK.Management of femoral head

osteonecrosis: Current concepts. IndianJ Orthop. 2015 Jan-Feb;49(1):28-45.

3, Vardhan H, Tripathy SK, Sen RK,Aggarwal S, Goyal T. EpidemiologicalProfile of Femoral Head Osteonecrosis inthe North Indian Population. Indian JOrthop.2018 Mar-Apr;52(2):140-146.

4. Gayana S, Bhattacharya A, Sen RK,Singh P, Prakash M, Mittal BR. F-18fluoride positron emission tomography/computed tomography in the diagnosis ofavascular necrosis of the femoral head:Comparison with magnetic resonanceimaging. Indian J Nucl Med. 2016 Jan-Mar;31(1):3-8.

5. Agrawal K, Tripathy SK, Sen RK,Santhosh S, Bhattacharya A. Nuclearmedicine imaging in osteonecrosis of hip:Old and current concepts. World J Orthop.2017 Oct 18;8(10):747-753.

6. Deivaraju C, Tripathy SK, Sen RK,Aggarwal S Efficacy of CoreDecompression in PosttraumaticOsteonecrosis of HipJ Hip Surg 2017;01(01): 019-021

7. Hernigou P, Beaujean F. Treatment ofosteonecrosis with autologous bonemarrow grafting. Clin Orthop Relat Res.2002;405:14–23

8. Gangji V, Hauzeur JP, Matos C, DeMaertelaer V, Toungouz M, LambermontM. Treatment of osteonecrosis of thefemoral head with implantation ofautologous bone-marrow cells. A pilotstudy. J Bone Joint Surg Am. 2004; 86-a(6):1153–60

101

9. Andriolo L, Merli G, Tobar C, AltamuraSA, Kon E, Filardo G. Regenerativetherapies increase survivorship ofavascular necrosis of the femoral head: asystematic review and meta-analysis. IntOrthop. 2018;6

10. Gangji V, De Maertelaer V, HauzeurJP. Autologous bone marrow cellimplantation in the treatment of non-traumatic osteonecrosis of the femoralhead: Five year follow-up of a prospectivecontrolled study. Bone. 2011;49(5):1005–9

11. Zhao D, Cui D, Wang B, Tian F, GuoL, Yang L, et al. Treatment of early stageosteonecrosis of the femoral head withautologous implantation of bone marrow-derived and cultured mesenchymal stemcells. Bone. 2012; 50(1):325–30

12. Sen RK, Tripathy SK, Aggarwal S,Marwaha N, Sharma RR, Khandelwal N.Early results of core decompression andautologous bone marrow mononuclearcells instillation in femoral headosteonecrosis: a randomized controlstudy. J Arthroplast. 2012;27(5): 679–86

13. Rastogi S, Sankineani SR, Nag HL,Mohanty S, Shivanand G, Marimuthu K,et al. Intralesional autologousmesenchymal stem cells in managementof osteonecrosis of femur: a preliminarystudy. Musculoskelet Surg. 2013;97(3):223–8

14. Ma Y, Wang T, Liao J, Gu H, Lin X,Jiang Q, et al. Efficacy of autologous bonemarrow buffy coat grafting combined with

core decompression in patients withavascular necrosis of femoral head: aprospective, double-blinded, randomized,controlled study. Stem Cell Res Ther.2014;5(5):115

15. Mao Q, Wang W, Xu T, Zhang S, XiaoL, Chen D, et al. Combination treatmentof biomechanical support and targetedintra-arterial infusion of peripheral bloodstem cells mobilized by granulocyte-colony stimulating factor for theosteonecrosis of the femoral head: arandomized controlled clinical trial. J BoneMiner Res. 2015;30(4):647–56 .

16. Tabatabaee RM, Saberi S, Parvizi J,Mortazavi SM, Farzan M. CombiningConcentrated Autologous Bone MarrowStem Cells Injection With CoreDecompression Improves Outcome forPatients with Early-Stage Osteonecrosisof the Femoral Head: A ComparativeStudy. J Arthroplast. 2015;30(9 Suppl):11–5

17. Gangji V, Hauzeur JP, Matos C, DeMaertelaer V, Toungouz M, LambermontM. Treatment of osteonecrosis of thefemoral head with implantation ofautologous bone-marrow cells. A pilotstudy. J Bone Joint Surg Am. 2004; 86-a(6):1153–60

18. Pepke W, Kasten P, Beckmann NA,Janicki P, Egermann M. CoreDecompression and Autologous BoneMarrow Concentrate for Treatment ofFemoral Head Osteonecrosis: ARandomized Prospective Study. Orthop

102 Orthobiologics

Rev (Pavia). 2016;8(1):6162

19. Gianakos AL, Moya-Angeler J, DuggalS, Zambrana L, Fields KG, Mintz DN, etal. The Efficacy of Bisphosphonates withCore Decompression and MesenchymalStem Cells Compared withBisphosphonates Alone in the Treatmentof Osteonecrosis of the Hip: aRetrospective Study. HSS J. 2016; 12(2):137–44

20. Lim YW, Kim YS, Lee JW, Kwon SY.Stem cell implantation for osteonecrosisof the femoral head. Exp Mol Med.2013;45:e61

21. Papakostidis C, Tosounidis TH, JonesE, Giannoudis PV. The role of “celltherapy” in osteonecrosis of the femoralhead. A systematic review of the literatureand meta-analysis of 7 studies. ActaOrthop. 2016;87(1):72–8

22. Hauzeur JP, De Maertelaer V,Baudoux E, Malaise M, Beguin Y, GangjiV. Inefficacy of autologous bone marrowconcentrate in stage three osteonecrosis:a randomized controlled double-blind trial.Int Orthop. 2017; 7:1429–35

23. Houdek MT, Wyles CC, Collins MS,Howe BM, Terzic A, Behfar A, et al. StemCells Combined With Platelet-rich PlasmaEffectively Treat Corticosteroid-inducedOsteonecrosis of the Hip: A ProspectiveStudy. Clin Orthop Relat Res.2018;476(2):388–97

24. Choudhery MS, Badowski M, MuiseA, Pierce J, Harris DT. Donor age

negatively impacts adipose tissue-derivedmesenchymal stem cell expansion anddifferentiation. J Transl Med. 2014;12:8 .

25. D’Ippolito G, Schiller PC, Ricordi C,Roos BA, Howard GA. Age-relatedosteogenic potential of mesenchymalstromal stem cells from human vertebralbone marrow. J Bone Miner Res.1999;14(7):1115–22

26. Stenderup K, Justesen J, Clausen C,Kassem M. Aging is associated withdecreased maximal life span andaccelerated senescence of bone marrowstromal cells. Bone. 2003;33(6):919–26 .

27. Aksu AE, Rubin JP, Dudas JR, MarraKG. Role of gender and anatomical regionon induction of osteogenic differentiationof human adipose-derived stem cells. AnnPlast Surg. 2008;60(3):306–22.

26. Stenderup K, Justesen J, Clausen C,Kassem M. Aging is associated withdecreased maximal life span andaccelerated senescence of bone marrowstromal cells. Bone. 2003;33(6):919–26 .

27. Aksu AE, Rubin JP, Dudas JR, MarraKG. Role of gender and anatomical regionon induction of osteogenic differentiationof human adipose-derived stem cells. AnnPlast Surg. 2008;60(3):306–22.

28. Baer PC, Geiger H. Adipose-derivedmesenchymal stromal/stem cells: tissuelocalization, characterization, andheterogeneity. Stem Cells Int. 2012;2012:812693

29. Wyles CC, Houdek MT, Crespo-Diaz

103

RJ, Norambuena GA, Stalboerger PG,Terzic A, et al. Adipose-derivedMesenchymal Stem Cells ArePhenotypically Superior for Regenerationin the Setting of Osteonecrosis of theFemoral Head. Clin Orthop Relat Res.2015;473(10):3080–90.

30. Lee JS, Lee JS, Roh HL, Kim CH, JungJS, Suh KT. Alterations in thedifferentiation ability of mesenchymal stemcells in patients with nontraumaticosteonecrosis of the femoral head:comparative analysis according to the riskfactor. J Orthop Res. 2006;24(4):604–9.

31. Suh KT, Kim SW, Roh HL, Youn MS,Jung JS. Decreased osteogenicdifferentiation of mesenchymal stem cellsin alcohol-induced osteonecrosis. ClinOrthop Relat Res. 2005;431:220–5

32. Houdek MT, Wyles CC, Packard BD,Terzic A, Behfar A, Sierra RJ. DecreasedOsteogenic Activity of Mesenchymal StemCells in Patients With Corticosteroid-Induced Osteonecrosis of the FemoralHead. J Arthroplast. 2016;31(4):893–8

33. Wang BL, Sun W, Shi ZC, Lou JN,Zhang NF, Shi SH, et al. Decreasedproliferation of mesenchymal stem cellsin corticosteroid-induced osteonecrosis offemoral head. Orthopedics.2008;31(5):444

34. Lee HJ, Kang KS, Kang SY, Kim HS,Park SJ, Lee SY, et al. Immunologicproperties of differentiated andundifferentiated mesenchymal stem cellsderived from umbilical cord blood. J Vet

Sci. 2015;17(3):289–97.

35. Cai J, Wu Z, Huang L, Chen J, Wu C,Wang S, et al. Cotransplantation of bonemarrow mononuclear cells and umbilicalcord mesenchymal stemcells in avascularnecrosis of the femoral head. TransplantProc. 2014;46(1): 151–5

36. Chen C, Qu Z, Yin X, Shang C, Ao Q,Gu Y, et al. Efficacy of umbilical cord-derived mesenchymal stem cell-basedtherapy for osteonecrosis of the femoralhead: A three-year follow-up study. MolMed Rep. 2016;14(5):4209–15

37. Muschler GF, Boehm C, Easley K.Aspiration to obtain osteoblast progenitorcells from human bone marrow: theinfluence of aspiration volume. J BoneJoint Surg Am. 1997;79(11):1699–709

38. Hernigou P, Poignard A, Beaujean F,Rouard H. Percutaneous autologousbone-marrow grafting for nonunions.Influence of the number and concentrationof progenitor cells. J Bone Joint Surg Am.2005;87(7):1430–7

39. Hernigou P, Trousselier M, RoubineauF, Bouthors C, Chevallier N, Rouard H, etal. Stem Cell Therapy for the Treatmentof Hip Osteonecrosis: A 30-Year Reviewof Progress. Clin Orthop Surg.2016;8(1):1–8

40. Hernigou P, Poignard A, Zilber S,Rouard H. Cell therapy of hiposteonecrosis with autologous bonemarrow grafting. Indian J Orthop.2009;43(1):40–5

104 Orthobiologics

41. Aarvold A, Smith JO, Tayton ER, JonesAM, Dawson JI, Lanham S, et al. Frombench to clinic and back: skeletal stemcells and impaction bone grafting forregeneration of bone defects. J Tissue EngRegen Med. 2014;8(10):779–86

42. Aarvold A, Smith JO, Tayton ER, JonesAM, Dawson JI, Lanham S, et al. A tissueengineering strategy for the treatment ofavascular necrosis of the femoral head.Surgeon. 2013;11(6):319–25

43. Kang JS, Moon KH, Kim BS, KwonDG, Shin SH, Shin BK, et al. Clinicalresults of auto-iliac cancellous bone graftscombined with implantation of autologousbone marrow cells for osteonecrosis of thefemoral head: a minimum 5-year follow-up. Yonsei Med J. 2013;54(2):510–5

44. Aoyama T, Goto K, Kakinoki R,Ikeguchi R, Ueda M, Kasai Y, et al. Anexploratory clinical trial for idiopathicosteonecrosis of femoral head by culturedautologous multipotent mesenchymalstromal cells augmented with vascularizedbone grafts. Tissue Eng B Rev.2014;20(4):233–42

45. Zhao D, Liu B, Wang B, Yang L, XieH, Huang S, et al. Autologous bonemarrow mesenchymal stem cellsassociated with tantalum rod implantationand vascularized iliac grafting for thetreatment of end-stage osteonecrosis ofthe femoral head. Biomed Res Int.2015;240506

46. Yamasaki T, Yasunaga Y, Ishikawa M,Hamaki T, Ochi M. Bone-marrow-derivedmononuclear cells with a poroushydroxyapatite scaffold for the treatmentof osteonecrosis of the femoral head: apreliminary study. J Bone Joint Surg Br.2010;92(3):337–41

47. Liu Y, Liu S, Su X. Coredecompression and implantation of bonemarrow mononuclear cells with poroushydroxylapatite composite filler for thetreatment of osteonecrosis of the femoralhead. Arch Orthop Trauma Surg.2013;133(1):125–33

48. Pak J. Autologous adipose tissue-derived stem cells induce persistent bone-like tissue in osteonecrotic femoral heads.Pain physician. 2012;15(1):75–85

49. Pilge H, Bittersohl B, SchneppendahlJ, Hesper T, Zilkens C, Ruppert M, et al.Bone Marrow Aspirate Concentrate inCombination With Intravenous IloprostIncreases Bone Healing in Patients WithAvascular Necrosis of the Femoral Head:A Matched Pair Analysis. Orthop Rev.2016;8(4):6902

Orthobiologics in the treatment of EarlyOsteoarthritis - A Systematic Review

1. Schminke B, Miosge N. Cartilage repairin vivo: the role of migratory progenitorcells. Curr Rheumatol Rep. 2014Nov;16(11):461.

2. Allen KD, Golightly YM. State of the

105

evidence. Curr Opin Rheumatol.2015;27(3):276-83.

3. Kon E, Filardo G, Drobnic M, Madry H,Jelic M, et al. (2012) Nonsurgicalmanagement of early knee osteoarthritis.Knee Surg Sports Traumatol Arthrosc 20:436-449

4. Aggarwal A (2015) Emergence ofOrthobiologics as a Novel TherapeuticModality for Osteoarthritis of Knee.Rheumatology (Sunnyvale) 5:159. doi:10.4172/2161-1149.1000159

5. Arnoczky SP, Fu FH, Mishra AK.Platelet-rich plasma: Clarifying the issues.JAAOS, Volume 18, Issue 7, July 2010.

6. Margolis DJ, Kantor J, Santanna J,Strom BL, Berlin JA. Effectiveness ofPlatelet Releasate for the Treatment ofDiabetic Neuropathic Foot Ulcers.Diabetes Care 2001 Mar; 24(3): 483-488.

7. Marx RE. Platelet-rich plasma: evidenceto support its use. J Oral Maxillofac Surg.2004 Apr;62(4):489-96.

8. Sampson S, Reed M, Silvers H, MengM, Mandelbaum B (2010) Injection ofplatelet-rich plasma in patients withprimary and secondary kneeosteoarthritis: a pilot study. Am J Phys MedRehabil 89: 961-969.

9. Wang-Saegusa A, Cugat R, Ares O,Seijas R, Cuscó X, et al. (2011) Infiltrationof plasma rich in growth factors for

osteoarthritis of the knee short-terme‡ects on function and quality of life. ArchOrthop Trauma Surg 131: 311-317. 13.

10. Kon E, Mandelbaum B, Buda R,Filardo G, Delcogliano M, et al. (2011)Platelet-rich plasma intra-articularinjection versus hyaluronic acidviscosupplementation as treatments forcartilage pathology: from earlydegeneration to osteoarthritis.Arthroscopy 27: 1490-1501.

11. Spaková T, Rosocha J, Lacko M,Harvanová D, Gharaibeh A (2012)Treatment of knee joint osteoarthritis withautologous platelet-rich plasma incomparison with hyaluronic acid. Am JPhys Med Rehabil 91: 411-417.

12. Sánchez M, Fiz N, Azofra J, UsabiagaJ, Aduriz Recalde E, et al. (2012) Arandomized clinical trial evaluating plasmarich in growth factors (PRGFEndoret)versus hyaluronic acid in the short-termtreatment of symptomatic kneeosteoarthritis. Arthroscopy 28: 1070-1078.

13. Anitua E, Sánchez M, Aguirre JJ,Prado R, Padilla S, et al. (2014) Efficacyand safety of plasma rich in growth factorsintra-articular infiltrations in the treatmentof knee osteoarthritis. Arthroscopy 30:1006-1017.

14. Dai WL, Zhou AG, Zhang H, Zhang J.Efficacy of Platelet-Rich Plasma in theTreatment of Knee Osteoarthritis: A Meta-

106 Orthobiologics

analysis of Randomized Controlled Trials.Arthroscopy. 2017 Mar;33(3):659-670.e1.doi: 10.1016/j.arthro.2016.09.024.

15. Martini LI, Via AG, Fossati C, RandelliF, Randelli P, Cucchi D. Single Platelet-Rich Plasma Injection for Early Stage ofOsteoarthritis of the Knee. Joints. 2017Jun 5;5(1):2-6. doi: 10.1055/s-0037-1601405.

16. Di Martino A, Di Matteo B, Papio T,Tentoni F, Selleri F, Cenacchi A, Kon E,Filardo G. Platelet-Rich Plasma VersusHyaluronic Acid Injections for theTreatment of Knee Osteoarthritis: Resultsat 5 Years of a Double-Blind, RandomizedControlled Trial. Am J Sports Med. 2018Dec 13:363546518814532.

17. Wakitani S, Imoto K, Yamamoto T,Saito M, Murata N, et al. (2002) Humanautologous culture expanded bonemarrow mesenchymal cell transplantationfor repair of cartilage defects inosteoarthritic knees. OsteoarthritisCartilage 10: 199-206.

18. Centeno CJ, Busse D, Kisiday J,Keohan C, Freeman M, et al. (2008)Increased knee cartilage volume indegenerative joint disease usingpercutaneously implanted, autologousmesenchymal stem cells. Pain Physician11: 343-353.

19. Emadedin M, Aghdami N, Taghiyar L,Fazeli R, Moghadasali R, et al. (2012)Intra-articular injection of autologous

mesenchymal stem cells in six patientswith knee osteoarthritis. Arch Iran Med 15:422-428.

20. Orozco L, Munar A, Soler R, AlbercaM, Soler F, et al. (2013) Treatment of kneeosteoarthritis with autologousmesenchymal stem cells: a pilot study.Transplantation 95: 1535-1541.

21. Kim JD, Lee GW, Jung GH, Kim CK,Kim T, et al. (2014) Clinical outcome ofautologous bone marrow aspiratesconcentrate (BMAC) injection indegenerative arthritis of the knee. Eur JOrthop Surg Traumatol 24: 1505-1511.

22. Chahla J, Dean CS, Moatshe G,Pascual-Garrido C, Serra Cruz R,LaPrade RF. Concentrated Bone MarrowAspirate for the Treatment of ChondralInjuries and Osteoarthritis of the Knee: ASystematic Review of Outcomes. OrthopJ Sports Med. 2016 Jan 13;4(1):2325967115625481.

23. Moatshe G, Morris ER, Cinque ME,Pascual-Garrido C, Chahla J,Engebretsen L, Laprade RF. Biologicaltreatment of the knee with platelet-richplasma or bone marrow aspirateconcentrates. Acta Orthop. 2017Dec;88(6):670-674. doi: 10.1080/17453674.2017.1368899.

24. Themistocleous GS, Chloros GD,Kyrantzoulis IM, Georgokostas IA,Themistocleous MS, Papagelopoulos PJ,Savvidou OD. Effectiveness of a single

107

intra-articular bone marrow aspirateconcentrate (BMAC) injection in patientswith grade 3 and 4 knee osteoarthritis.Heliyon. 2018 Oct 18;4(10):e00871.

25. Bentley G, Minas T. Treating jointdamage in young people. BMJ.2000;320:1585–1588.

26. Gooding CR, Bartlett W, Bentley G,Skinner JA, Carrington R, Flanagan A. Aprospective, randomised study comparingtwo techniques of autologous chondrocyteimplantation for osteochondral defects inthe knee: Periosteum covered versus typeI/III collagen covered. Knee. 2006;13:203–210.

27. Basad E, Ishaque B, Bachmann G,Stürz H, Steinmeyer J: Matrix-inducedautologous chondrocyte implantationversus microfracture in the treatment ofcartilage defects of the knee: a 2-yearrandomised study. Knee Surg SportsTraumatol Arthrosc 2010, 18:519-527.

28. Erggelet C, Sittinger M, Lahm A: Thearthroscopic implantation of autologouschondrocytes for the treatment of fullthickness cartilage defects of the kneejoint. Arthroscopy 2003, 19:108-110

29. Della Villa S, Kon E, Filardo G, RicciM, Vincentelli F, Delcogliano M, MarcacciM: Does intensive rehabilitation permitearly return to sport without compromisingthe clinical outcome after arthroscopicautologous chondrocyte implantation inhighly competitive athletes? Am J Sports

Med 2010, 38:68-77.

30. Choi et al., Gel-type autologouschondrocyte (Chondron™) implantationfor treatment of articular cartilage defectsof the knee BMC MusculoskeletalDisorders 2010, 11:103

31. Peterson, L., Vasiliadis, H. S.,Brittberg, M., & Lindahl, A. (2010).Autologous Chondrocyte Implantation: ALong-term Follow-up. The AmericanJournal of Sports Medicine, 38(6), 1117–1124.

32. A. Viste, M. Piperno, R. Desmarchelier,S. Grosclaude, B. Moyen, M.H. Fessy,Autologous chondrocyte implantation fortraumatic full-thickness cartilage defectsof the knee in 14 patients: 6-yearfunctional outcomes, Orthop. Traumatol.Surg. Res. 98 (2012) 737–743,

33. Minas T, Von Keudell A, Bryant T,Gomoll AH. The John Insall Award: Aminimum 10-year outcome study ofautologous chondrocyte implantation. ClinOrthop Relat Res. 2013;472(1):41-51.

34. Mistry H, Connock M, Pink J, et al.Autologous chondrocyte implantation inthe knee: systematic review and economicevaluation. Southampton (UK): NIHRJournals Library; 2017 Feb.

35. Jungmann, P.M., Gersing, A.S.,Baumann, F. et al. Knee Surg SportsTraumatol Arthrosc (2018).

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