1

Anti Ageing ConferenceLondon

9-11 September, 2010

Self-Repair in Degenerative joint

DiseaseValerio Di Nicola M.D. Ph.D. and Renato Di Nicola Valerio Di Nicola M.D. Ph.D. and Renato Di Nicola

M.D.M.D.✟✟

Center for Applied Clinical Researchof Degenerative Arthropaties - Rome, Italy

andInterbion Foundation

for Basis Biomedical Research - Gordola, Switzerland

Objective of The Study

Stimulate the local Innate Stem-Cellsin order induce tissue repair

Osteoarthritis of Hip and KneeOsteoarthritis of Hip and Knee

Regenerative MedicineHistory

Davis JW, Davis JW, Johns Hopkins Med J Johns Hopkins Med J 19101910

Regenerative MedicineHistory

Filatov VP, Kirurgiia Filatov VP, Kirurgiia 19551955

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Regenerative MedicineHistory

Cerletti U, Ann NY Acad Sci Cerletti U, Ann NY Acad Sci 19861986

Vishwakarma and Khare

used Amniotic non viable Membranes

to performe Arthroplastics of the Hip

(Vishwakarma GK, J Bone Joint Surg 1986)(Vishwakarma GK, J Bone Joint Surg 1986)

Back in 1960’s we began to useamniotic non viable membranes

(Amniex®)

Amnios was inserted in periarticular Amnios was inserted in periarticular spacespace

to treat Symptomatic Arthritisto treat Symptomatic Arthritis

Extra cellular Microenvironment and Tissue

Renewal• Platelet-Fibronectin complexes (Bianchini P et all, Int J of Tissue React 1981)

• Cellular death, Cell lyses and extra cellular Nucleic Acids diffusion, localregenerative stimulus(Gailit J, Curr Opin Cell Biol 1994)

• Nucleic Acids growth promoters for fibroblasts, osteoblasts and endothelial cellsin vitro and in vivo(Joyce ME et all Prog Clin Biol Res 1991; Henning UUG et all, Mutation

Research1996; Bowler WB et all, Bone 2001)

Role of Extra cellular Nucleic Acids

Extra cellular Microenvironment and Tissue

RenewalRole of Extra cellular Nucleic Acids• Extra cellular Polydeoxyribonucleotides ( Pdrn) stimulate synthesis Nucleic Acids

through the salvage pathways (P1[A1]-P2 receptors) on fibroblasts andosteoblasts cultures ( Thellung S et all, Life Sciences 1999; Nakamura E et all, Am J Phisiol Cell

Phisiol 2000)

• Synergistic role between GF (TGF-beta1, FGF2, etc) and Pdrn(Ding GJI, Biochem Biophys Res Commun 1990; Chavan AJ, Biochemistry

1994)

• Pdrn promotes osteoblasts growth in cultures(Guizzardi S et all, Life Sciences 2003)

• Bone regeneration in rat through heat deproteinate bone matrix and Pdrn(Guizzardi S et all, Micron 2007)

Extra cellular Microenvironment and Tissue

RenewalRole of Trauma Necrosis and Hypoxia

Surgical TraumaSurgical Trauma

NecrosisNecrosis HypoxiaHypoxia

locallocal

G FG FVEGF, FGF 1VEGF, FGF 1--2, TGF beta 12, TGF beta 1

(Abdollahi H et all, J Surg Res 2009)(Abdollahi H et all, J Surg Res 2009)

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Extra cellular Microenvironment and Tissue

RenewalRole of Trauma Necrosis and Hypoxia

•• TGF beta 1TGF beta 1Inhibits metalloproteinases synthesis Inhibits metalloproteinases synthesis Promotes proliferation articular chondrocytes and nucleus polpusPromotes proliferation articular chondrocytes and nucleus polpusus us

cellscells(Nakai T et all, Arthritis Res Ther 2008 )

• FGF 1FGF 1--22Increases proliferation Multipotent Mesenchymal stem cells (MSCsIncreases proliferation Multipotent Mesenchymal stem cells (MSCs))(Rider DA et all, Stem Cells 2008 )

• HIF alphaHIF alphaProduces effects over stem cells differentiation status Produces effects over stem cells differentiation status (Moreno MV et all, Journal Biol Chem 2010 )

Extra cellular Microenvironment and Tissue

RenewalRole of Heat Shock Proteins (HSP)

(Wu C, Annu Rev Cell Dev Biol 1995 (Wu C, Annu Rev Cell Dev Biol 1995 -- De Maio A, Shock 1999 De Maio A, Shock 1999 –– Santoro MG, Biochem Santoro MG, Biochem Pharmacol 2000)Pharmacol 2000)

QuickTime™ and aH.264 decompressor

are needed to see this picture.

QuickTime™ and aH.264 decompressor

are needed to see this picture.

Cellular stress ResponseHSP

Cellular stress ResponseCellular stress ResponseHSPHSP

High temperature or High temperature or other physical, chemical other physical, chemical

ororbiological stressbiological stress

Extra cellular Microenvironment and Tissue

RenewalRole of Heat Shock Proteins (HSP)

HSP Functions in Differentiated Cells HSP Functions in Differentiated Cells • Chaperones for other proteinsChaperones for other proteins• HousekeepingHousekeeping•• Immunity Immunity

HSP Functions in Stem Cells HSP Functions in Stem Cells •• High levels of chaperone expression increase stem cells stress High levels of chaperone expression increase stem cells stress

tolerance and self renewaltolerance and self renewal•• Hsp20 protects MSCs against cell death by oxidative stressHsp20 protects MSCs against cell death by oxidative stress

(Pierpaoli EV, Ann NY Acad Sci 2005 (Pierpaoli EV, Ann NY Acad Sci 2005 -- Prisloo E et all, Bioessays 2009 Prisloo E et all, Bioessays 2009 –– Wang X et all, Wang X et all, Stem Cells 2009 )Stem Cells 2009 )

Our preliminaryClinical Experience

Pdrn alonePdrn alone

Blood+PdrnBlood+Pdrn

68%

43%

40% fewer Infiltrations40% fewer Infiltrations30% fewer No Responders30% fewer No Responders

Patients

Patients

We devised a new biomaterial

Chemical and Physical Compatibility Chemical and Physical Compatibility Chemical and Physical Compatibility

Long Absorption TimeLong Absorption TimeLong Absorption Time

Unified effects between Pdrn, HSP and local GFUnified effects between Pdrn, HSP and local GFUnified effects between Pdrn, HSP and local GF

No Toxicity Nor Side EffectsNo Toxicity Nor Side EffectsNo Toxicity Nor Side Effects

GelGel--RepairerRepairer

with the following properties

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GelGel--RepairerRepairer✴✴

is a jellified mixture made with distressed, processed Blood, Pdrn and thickening

substance

Mechanism of action hypothesisMechanism of action hypothesis•• Prolonged stimulating action by Pdrn and Prolonged stimulating action by Pdrn and

HPS on innate local Stem CellsHPS on innate local Stem Cells•• Scaffold for activated Stem CellsScaffold for activated Stem Cells

✴✴patent pending RM2009A000485patent pending RM2009A000485

GelGel--RepairerRepairerEffects on Joint Tissue

Fibroblasts activation inducing synthesis of elastin Fibroblasts activation inducing synthesis of elastin and collagen type II, resulting increase in flexibility and collagen type II, resulting increase in flexibility and compliance of joint capsule with reduction of and compliance of joint capsule with reduction of intraintra--articular pressure. articular pressure.

Stimulation on periosteal tissue and osteoblast Stimulation on periosteal tissue and osteoblast cells inducing proliferation and bone repaircells inducing proliferation and bone repair

Generally xGenerally x--rays show increase of cartilaginous rays show increase of cartilaginous matrix layer matrix layer

Material and MethodsFrom January 2003 until June 2009 we have treated 948 From January 2003 until June 2009 we have treated 948 patients with DJD covering virtually all medium and large patients with DJD covering virtually all medium and large articulations articulations

The focus of this analysis is on two Groups of patientsThe focus of this analysis is on two Groups of patients

Group 1 was composed of 86 over eighties patients Group 1 was composed of 86 over eighties patients affected by DJD of the hip or/and kneeaffected by DJD of the hip or/and knee

Group II of 90 patients around fifty years old were affected Group II of 90 patients around fifty years old were affected by the same disease but the causes of DJD were different as by the same disease but the causes of DJD were different as postpost--traumatic, congenital hip dysplasia, arthritis induced by traumatic, congenital hip dysplasia, arthritis induced by severe postural defectssevere postural defects

Material and Methods

The patients in Group 1 were judged high surgical risk for The patients in Group 1 were judged high surgical risk for prosthesis (the most were ASA III prosthesis (the most were ASA III -- IV)IV)

Group 1 and Group II had noGroup 1 and Group II had no--responders to currently responders to currently adopted conservative therapiesadopted conservative therapies

All patients gave informed consent before entering the studyAll patients gave informed consent before entering the study

Patients who underwent corticosteroids therapy over the last Patients who underwent corticosteroids therapy over the last month, INR over 3.5 and affected by acute rheumatic diseases month, INR over 3.5 and affected by acute rheumatic diseases were excludedwere excluded

FollowFollow--ups: were in short term ( 6 ups: were in short term ( 6 –– 12 months ) and long term 12 months ) and long term ( 24 ( 24 –– 48 months )48 months )

•• The treatment was performed simultaneously on 2 or 3 areas of The treatment was performed simultaneously on 2 or 3 areas of the joint previouslythe joint previouslyevaluated by clinical and radiological assessmentevaluated by clinical and radiological assessment

•• The treatment was repeated at weekly intervals. Preliminary The treatment was repeated at weekly intervals. Preliminary results were assessedresults were assessedafter three proceduresafter three procedures

Material and MethodsGelGel--Repairer Repairer ProcedureProcedure

Clinical Assessment QuestionnaireClinical Assessment Questionnaire

•• WOMAC (Western Ontario and McMaster Universities) for HipWOMAC (Western Ontario and McMaster Universities) for Hipand Kneeand Knee

•• Harris Hip ScoreHarris Hip Score•• Knee Society Score and Functional scoreKnee Society Score and Functional score

Radiological Assessment (XRadiological Assessment (X--Ray) Ray)

•• Kellgren and Lawrence Scale (K&L) Kellgren and Lawrence Scale (K&L)

Material and MethodsCriteria for evaluating resultsCriteria for evaluating results

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ResultsStatistical analysis was performed by Simona Dinicola Ph.D. of Statistical analysis was performed by Simona Dinicola Ph.D. of

the Experimental Medicine Department the Experimental Medicine Department ““SapienzaSapienza”” University University of Rome.of Rome.

Data was expressed as means of Data was expressed as means of ±± SD and a statistical analysis SD and a statistical analysis was performed through Studentwas performed through Student’’s T test. Differences were s T test. Differences were considered significant at the level of p<0.01. The statistical considered significant at the level of p<0.01. The statistical analysis was performed by using GraphPad Instat software analysis was performed by using GraphPad Instat software

(GraphPad Software, Inc; San Diego, CA, USA).(GraphPad Software, Inc; San Diego, CA, USA).

Results

Average Age (y) 83

Males/Females 37/49

Joint Treated hip/knee

Patients (n=86); Lost* (n=12)

Patients’ Clinical Features Group 1Patients’ Clinical Features Group 1

Patients (n=90); Lost* (n=10)

Patients (n=86); Lost* (n=12) Patients (n=90); Lost* (n=10)

Not Responsive 5

Follow Up Lost 5

Died during treat. 2

Average Age (y) 51

Males/Females 39/51

Joint Treated hip/knee

Not Responsive 8

Follow Up Lost 2

Died during treat. 0

Lost Patients Group 1Lost Patients Group 1 Lost Patients Group IILost Patients Group II

Patients’ Clinical Features Group IIPatients’ Clinical Features Group II

From January 2003 to June 2009 we have treated two Groups of

Patients affected by DJD of the hip or / and knee

* Lost patients: they died during the treatment; they did not re* Lost patients: they died during the treatment; they did not respond to the treatment; their follow up was spond to the treatment; their follow up was missedmissed

ResultsAverage clinical baseline assessment and classification before and after

treatment Average

Basic Score aa

AverageShort Term Score

bb

AverageLong Term Score ccClassifications

31.3 75.2 73.7

22.5 64.4 63.6

35.4 76 74.8

25.1 54.4 53.7

WOMAC

Harris Hip

Knee Society

Knee Society Function

AverageBasic Score aa

AverageShort Term Score

bb

AverageLong Term Score ccClassifications

31 78.5 75.5

22 67 65

36 76 75

25 60 58

WOMAC

Harris Hip

Knee Society

Knee Society Function

Group IIGroup II

Group 1Group 1

aa basic score: before treatment; basic score: before treatment; bb shortshort--term score: within 6term score: within 6--12 months of treatment; 12 months of treatment; cc longlong--term score: within 24term score: within 24--48 months of 48 months of treatmenttreatment

ResultsKellgren and Lawrence scale (K&L)

Basic Score aa Short Term Scorebb Long Term Score cc

Basic Score aa Short Term Scorebb Long Term Score cc

Group IIGroup II

Group 1Group 1

aa basic score: before treatment; basic score: before treatment; bb shortshort--term score: within 6term score: within 6--12 months of treatment; 12 months of treatment; cc longlong--term score: within 24term score: within 24--48 months of 48 months of treatmenttreatment

54 patients GIII 7 patients GII 4 patients GII

32 patients GIV 23 patients GIII 14 patients GIII

50 patients GIII 21 patients GII 16 patients GII

30 patients GIV 20 patients GIII 14 patients GIII

Results

WOMAC hip score before and after treatment at short and long terWOMAC hip score before and after treatment at short and long termmData were expressed as mean Data were expressed as mean ±± SD. Differences were considered significant at the level SD. Differences were considered significant at the level of p<0.01of p<0.01■■ WW--bs = WOMAC basic scorebs = WOMAC basic score■■ Ws = WOMAC score (short / long term)Ws = WOMAC score (short / long term)

Results

WOMAC Knee score before and after treatment at short and long teWOMAC Knee score before and after treatment at short and long termrmData were expressed as mean Data were expressed as mean ±± SD. Differences were considered significant at the level SD. Differences were considered significant at the level of p<0.01of p<0.01■■ WW--bs = WOMAC basic scorebs = WOMAC basic score■■ Ws = WOMAC score (short / long term)Ws = WOMAC score (short / long term)

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Results

Harris Hip score before and after treatment at short and long teHarris Hip score before and after treatment at short and long termrmData were expressed as mean Data were expressed as mean ±± SDSDDifferences were considered significant at the level of p<0.01 Differences were considered significant at the level of p<0.01 ■■ HHHH--bs = Harris Hip basic scorebs = Harris Hip basic score■■ HHs = Harris Hip score (short / long term)HHs = Harris Hip score (short / long term)

Results

Knee Society score before and after treatment at short and long Knee Society score before and after treatment at short and long termtermData were expressed as mean Data were expressed as mean ±± SD. Differences were considered significant at the level SD. Differences were considered significant at the level of p<0.01 of p<0.01 ■■ KSKS--bs = Knee Society basic scorebs = Knee Society basic score■■ KSs = Knee Society score (short / long term)KSs = Knee Society score (short / long term)

Results

Knee Society function score before and after treatment at short Knee Society function score before and after treatment at short and long termand long termData were expressed as mean Data were expressed as mean ±± SD. Differences were considered significant at the level SD. Differences were considered significant at the level of p<0.01of p<0.01■■ KSKS--fbs = Knee Society function basic scorefbs = Knee Society function basic score■■ KS fs = Knee Society function score (short / long term)KS fs = Knee Society function score (short / long term)

Results

Correlation WOMAC classification versus Kellgren and Lawrence scCorrelation WOMAC classification versus Kellgren and Lawrence scale. Columns ale. Columns represent the average ratio Ws/Wrepresent the average ratio Ws/W--bs in respect to both downstaging and maintenance of bs in respect to both downstaging and maintenance of K&L scaleK&L scaleData were expressed as mean Data were expressed as mean ±± SD. Differences were considered significant at the level SD. Differences were considered significant at the level of p<0.05of p<0.05

WOMAC and K&L scale correlation

AmniosAmnios Placenta extractsPlacenta extracts

Discussion Discussion

Surgical Trauma NecrosisSurgical Trauma Necrosisand Hypoxiaand Hypoxia

BloodBlood++

PolydeoxyribonucleotidesPolydeoxyribonucleotides

HeatHeatStressStress

RegenerativeRegenerativeMicroenvironmentMicroenvironment

Stimulates Innate LocalStimulates Innate LocalStem CellsStem Cells

HSP + PdrnHSP + PdrnGelGel--RepairerRepairer

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Kellgren and Lawrence scaleshows a radiological down-

staging

Group 1Group 1 Group 1I

41%41% 51%51%

PatientsPatients PatientsPatientsRight hipRight hipA) IV grade K&L before treatmentA) IV grade K&L before treatmentB) III grade K&L after treatment (6th months followB) III grade K&L after treatment (6th months follow--

up)up)

Left KneeLeft KneeA) III grade K&L before treatmentA) III grade K&L before treatmentB) II grade K&L after treatment (20th months followB) II grade K&L after treatment (20th months follow--

up)up)

Right hipRight hipA) IV grade K&L before treatmentA) IV grade K&L before treatmentB) III grade K&L after treatment (43th months followB) III grade K&L after treatment (43th months follow--

up)up)

Right knee: severe chondrocalcinosis in DJDRight knee: severe chondrocalcinosis in DJDA) before treatmentA) before treatmentB) after treatment (6th months followB) after treatment (6th months follow--up). Mainly the up). Mainly the

medial medial compartment of the knee had been treatedcompartment of the knee had been treated

Left kneeLeft kneeA) III grade K&L before treatmentA) III grade K&L before treatmentB) II grade K&L after treatment (7th months followB) II grade K&L after treatment (7th months follow--

up)up)

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Right kneeRight kneeA) III grade K&L before treatmentA) III grade K&L before treatmentB) III grade K&L after treatment (8th months followB) III grade K&L after treatment (8th months follow--up)up)Clinical improvement was more evident than radiologicalClinical improvement was more evident than radiological

Kellgren and Lawrence scale is often Kellgren and Lawrence scale is often inadequateinadequate

to value structural joint modificationsto value structural joint modifications

Specificity and sensitivity of MR on soft tissue Specificity and sensitivity of MR on soft tissue providesprovides

better structural details increasing % of downbetter structural details increasing % of down--stagingstaging

RX vs MRbefore treatment 2005 after treatment 2010

Current limit of MR is the absence of Current limit of MR is the absence of standardizationstandardization

The histological interpretation of this radiological The histological interpretation of this radiological improvement still has to be accounted for improvement still has to be accounted for

RX vs MR

before treatment 2005 after treatment 2010

What is really causing clinical and radiological improvements in the treated

joint?

Our Data indicatesOur Data indicates

Pain

Biomechanics

Regenerative Regenerative MicroenvironmentMicroenvironment

Hypothesis about Repair Mechanism

Surgical Pocket filled by Surgical Pocket filled by GelGel--RepairerRepairer

Prolonged Prolonged RegenerativeRegenerative

Action over Stem Action over Stem CellsCells

Scaffold FunctionScaffold Function

Tissue Tissue RenewalRenewal

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Tissue RenewalProliferation and DifferentiativeProliferation and Differentiative

Stimulus on Fibroblasts and Stimulus on Fibroblasts and OsteoblastsOsteoblasts

Bone MatrixBone Matrix

ElastinElastin

Collagen Type IICollagen Type II

Tissue RenewalTGFTGF--beta1 promotes Condroblasts growthbeta1 promotes Condroblasts growth

FGF2 and HIF have effects on FGF2 and HIF have effects on MSCsMSCs

proliferation and differentiationproliferation and differentiation

Surgical Trauma Surgical Trauma GelGel--RepairerRepairer

GFGF Pdrn +HSPPdrn +HSP

Cartilage RegenerationCartilage Regeneration

Joint Structural Changeis Clinically confirmed by

Improved Articular FlexibilityImproved Articular FlexibilityImproved Articular Flexibility

Reduction Intra-articular PressureReduction IntraReduction Intra--articular Pressurearticular Pressure

Reduction of PainReduction of PainReduction of Pain

Increased Joint MotilityIncreased Joint MotilityIncreased Joint Motility

ConclusionCurrent Therapeutic Options in DJD

•• NSAIDNSAID’’s, steroids and analgesicss, steroids and analgesics

•• Local treatment such as ultrasound, laser, electrophoresis Local treatment such as ultrasound, laser, electrophoresis etcetc

•• Physiokinesis therapyPhysiokinesis therapy

•• Surgery (arthroscopy, prosthesis), Golden StandardSurgery (arthroscopy, prosthesis), Golden Standardfor advanced degenerative disease for advanced degenerative disease

ConclusionGelGel--RepairerRepairer

demonstrated a new valid option for DJD treatment

•• Minimally InvasiveMinimally Invasive

•• Patients acceptance and reliance on the procedurePatients acceptance and reliance on the procedure

•• The Therapy does not preclude additional therapiesThe Therapy does not preclude additional therapies

•• Simple Procedural Method, applicable on almost allSimple Procedural Method, applicable on almost allarticular areasarticular areas

•• Highly cost effectivenessHighly cost effectiveness

•• No collateral effects, no toxicity, no significant No collateral effects, no toxicity, no significant complicationscomplications

This study suggests a new methodological approach and This study suggests a new methodological approach and treatment of DJD based on tissue regeneration and treatment of DJD based on tissue regeneration and

restoration resulting in clinical resolution restoration resulting in clinical resolution

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Anti Ageing ConferenceLondon

9-11 September, 2010