recovery of function in anal incontinence after micro ... · anal incontinence is common and...

1

ABSTRACTAnal incontinence is common and significantlydiminishes quality of life. This work aims to in-vestigate the therapeutical potential of regener-ative lipofilling by lipoaspirated fat, washed andreduced in size by the new device Lipogems®, inpatients affected by chronic fecal incontinence.5 patients with fecal incontinence due to ob-

stetric injury and anorectal-pelvic surgery werefollowed up for 24 months after grafting of anaverage of 90 cc of aspirated and microfrag-mented fat (Lipogems®) in the external, internalanal sphincters and around pudendal nerves.Wexner-Incontinence-Score and Fecal-Inconti-nence-Quality-of-Life-Scale were used beforeand after treatment together with digital explo-ration, proctoscopy, endoanal ultrasound andanorectal manometry.All patients observed an improvement both

short and long term. The Wexner-Incontinence-Score improved from a preoperative mean of14.0 to 3.4 at 3 months after treatment and re-mained stable up to 24 months. Anorectalmanometry has reported over time significantimprovements of pressure both at rest and insqueeze. Ultrasonography showed reabsorptionof Lipogems tissue at 6 months and clear musclerepair at 12 and 24 months.Our preliminary results suggest that this re-

generative lipofilling can improve symptoms offecal incontinence due to muscular and neurallocal trauma. Further studies are necessary toclarify the underlying mechanism, but aparacrine action of the mesenchymal stem cellscontained in the fat tissue or in the purified fat

cells themselves is postulated, as at 6-12 monthswhen ecography of transplanted fat shows thatthe injected material disappeared while the ben-eficial clinical effects continue to be present andimprove over time.

Keywords: Fecal incontinence, Lesions of anal sphincters,Mesenchymal stem cells, Regenerative therapy, Lipogems.

INTRODUCTION

Fecal is a disabling condition with a strong psycho-social impact that affects 2% to 15% of the generalpopulation, although its true prevalence is certainlyunderestimated1,2. It is generally caused by a varietyof conditions and it often derives from a combina-tion of factors; nevertheless the most common ac-quired causes are lesions of anal sphincters, oftenassociated with damage to the peripheral innerva-tion, due to obstetric injury, trauma, and anorectal-pelvic surgery3-5. Treatment options of severeincontinence include surgical procedures likesphincteroplasty, stimulated graciloplasty and arti-ficial sphincter insertion6-10, while in mild andmedium incontinence are usually reserved conser-vative interventions such as drugs, pelvic floor mus-cle training, biofeedback, and sacral nervestimulation11-12. A minimally invasive alternativetreatment is the local injection of bulking agents toincrease the volume of sphincter apparatus. Thisprocedure, which employs biological or syntheticmaterials, improves the pressure of anal sphincter,but it is severely limited because of the absence ofdynamism and contractility of the materials whichare also subject to migration and resorption. How-

Recovery of Function in Anal Incontinence After Micro-Fragmented Fat Graft (Lipogems®) Injection: Two Years Follow Up of the First 5 Cases

A. Giori1, C. Tremolada2, R. Vailati1, S.E. Navone3, G. Marfia3, A.I. Caplan4

1Department of General Surgery, San Paolo University Hospital, Milan, Italy.2Istituto Image, Milan, Italy.3Laboratory of Experimental Neurosurgery and Cell Therapy, Neurosurgery Unit, Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy.

4Department of Biology, Skeletal Research Center, Case Western Reserve University, Cleveland, OH, USA.

A. Giori and C. Tremolada contributed equally to this work

CellR4 2015; 3 (2): e1544

Corresponding Author: Alberto Giori, MD; e-mail: [email protected]

2 A. Giori, C. Tremolada, R. Vailati, S.E. Navone, G. Marfia, A.I. Caplan MSCs, ADSCs express CD34 glycoprotein47, stro-mal markers and endothelial cell markers48. More-over, ADSCs have a higher yield upon isolation anda greater proliferative rate in culture when comparewith MSCs isolated from other sources49 and pos-sess restorative abilities that make them a good can-didate for regenerative therapy50.

The adipose tissue extracted from humans by agentle liposuction was treated through the use ofmechanical processes of microfracturing, filteringand washing in a complete closed system. The heal-ing potential of the MSCs contained in the adiposetissue is maintained by avoiding the use of enzymesand/or any other additives. Cellular biology studieshave revealed that Lipogems® product is composedof small adipose tissue spherical clusters withmicro-fragments of intact connective structuremaintained viable by vascular support, particularyrich in pericytes and MSCs exposed on the surfaceof the vascular stroma37,39. Several investigatorshave hypothesized that pericytes are the precursorsof the MSC, and their protruding position into theendothelial lumen of blood vessels makes themready to monitor and react to local or systemic sig-nals51-55. These properties contribute to make Li-pogems® able to survive in a tissue and facilitateengraftment when autologously inoculated in targettissues36.

MATERIALS AND METHODS

ETHICS STATEMENT AND SAMPLES COLLECTIONThe study was performed according to the Decla-ration of Helsinki. From June 2011, 5 consecutivepatients (4 females, 1 male) aged from 46 to 74(median 61 years), with at least 12 months historyof fecal incontinence to liquids and/or solid stoolconfirmed by incontinence diaries kept by patientsfor two week, were treated to correct fecal incon-tinence with injection of Lipogems® into anal re-gion. Fecal incontinence resulted from analsphincter injury, peripheral nerve damage, or acombination of both, as shown in Table 1. Aftertreatment, patients were followed-up for at least 2years. All patients were advised about the potentialbenefits and risks and gave informed consent tothe study approved by the local Ethics Committee.Before the Lipogems procedure, all patients haveattempted medical and rehabilitation treatmentswith unsatisfactory results. Moreover, oncologistshave provided a consent for the treatment in pa-tients with previous history of cancer.

ever, improvements in continence initially reportedwith the use of these techniques are not maintainedby long-term13-16.

Regenerative medicine is a great promise thatprovides hope for patients that display severe formsof degenerative or traumatic conditions, or for pa-tients who are refractory to conventional treat-ments17. In this context, mesenchymal stem cells(MSCs) have been extensively studied with regardto their potential therapeutic applications. MSCs aremultipotent adult stem cells capable of self-renewand differentiation potential, found in bone marrow(BM-MSCs) and in adipose tissue (ADSCs)18. Fur-thermore MSCs produce secreted factors such ascytokines and chemokines, or growth factors, whichmediate diverse functions by cross-talk between dif-ferent cell types, stimulating reparative re-sponses19,20.

Recent studies of regenerative medicine have re-vealed that BM-MSCs are able to stimulate the hostto differentiate into mature cells of various tis-sues21. In particular, BM-MSCs injected into skele-tal muscle generate favorable conditions for thegrowth of muscular cells by virtue of theirparacrine activity22-24.

Furthermore, recent experimental investigationsin vitro and in vivo have reported improved regen-eration of peripheral nerves with the use of BM-MSCs25-28. Because of encouraging use of MSCs torepair muscle damage in animal models with in-duced urinary29,30 and anal incontinence31,32, and, onthe basis of encouraging results by clinical studiesin patients with urinary33,34 and anal incontinence 35,we considered of employing microfractured andwashed adipose tissue with remarkable regenerativeproperties (Lipogems®)36-39 to repair lesions of theanal sphincters in adult patients with fecal inconti-nence. This new procedure, derived from lipofillingsurgical technique40-42, represents a new field of re-generative surgery whose therapeutic criteria arebased on the possibility of transferring the medici-nal cells that are normally present in several tissuesof adult human body, such as bone marrow, dentalpulp, and adipose tissue43-45, in those damaged areasof the human body that require repair.

Moreover MSCs isolated from adipose tissue,compared to bone marrow, present some peculiarcharacteristics. 1 g of adipose tissue contains 500times more MSCs than 1 g of bone marrow aspi-rate46. Indeed, besides showing phenotypic and tran-scriptional profiles similar to that of the other

3

DIAGNOSTIC PROCEDURESPrior to treatment, anal incontinence was evaluatedusing the Wexner Incontinence Score56 and the qual-ity of life was assessed with Fecal Incontinence Qual-ity of Life (FIQL) Scale57. Patients were alsosubjected to digital anorectal examination and wereinvestigated with proctoscopy and anorectal manom-etry (Medtronic Polygram – water perfused 4 chan-nel catheter). Furthermore, endoanal ultrasound wasperformed to evaluate lesions of the anal sphinctersusing a 10-MHz rotating endoprobe (Kretz ERW7-10AKP – Combison 401 GE Medical Systems). Li-pogems® treatment was conducted with patient placedin lithotomic position under general anesthesia, in asingle surgical session that includes 3 different steps:

1) Harvesting of adipose tissue from the patient; 2)Processing of adipose tissue with Lipogems® device;3) Re-inoculation of the product in the same patient.LIPOGEMS TREATMENT1. Harvesting of adipose tissue

The lower or the lateral abdomen were chosen asdonor sites for fat graft harvesting using a tech-nique derived from traditional surgical liposuction.A blunt 13G cannula with multiple elliptical holesof 1x2 mm distributed along it, was employed forfat suction. This disposable cannula, connectedwith a 10 cc syringe, allows a fast and a traumaticsuction of fat with few cannula strokes. Harvestingof fat is done 10 minutes after injection of salineand 1:500000 adrenaline in the selected area.

Table 1. Clinical features of the patients.

Patient History Wexner Endoanal Ano- Anorectal manometryscore ultrasonography cutaneous (values in mmHg)(0>20) reflex

A At the age of 27 difficult 11 Combined ruptures of EAS Normally Hypotonia at resting pressure, female birth with laceration and IAS localized in present low pressure increasingage 61 and episiotomy the anterior area of in squeeze

the muscular rings in Mean resting pressure = 14the middle and upper Maximum resting pressure = 30portion of anal canal Mean squeeze pressure = 33

Maximum squeeze pressure = 56B At the age of 55 anterior 14 Diffuse lesions of the EAS Impaired Hypotonia at resting pressure, male resection of the rectum and IAS with hypo-and low pressure increasing in age 61 with coloanal anastomosis, hyper-echogenic areas squeeze

followed by radiotherapy related to fibrous regression Mean resting pressure = 23and chemotherapy for Maximum resting pressure = 28rectal cancer Mean squeeze pressure = 31

Maximum squeeze pressure = 76C Aged 50 to 56 repeated 16 No evidence of injury Absent Resting pressure within normalfemale pelvic surgery and of the anal sphincters limits, low pressure increasingage 65 chemotherapy for in squeeze

ovarian carcinoma Mean resting pressure = 43Maximum resting pressure = 46Mean squeeze pressure = 55Maximum squeeze pressure = 84

D Sacral fracture in childhood. 13 Combined ruptures of EAS Normally Hypotonia at resting pressure, female At the age of 34 difficult and IAS localized in present low pressure increasing in age 46 birth with laceration and the anterior area of squeeze.

episiotomy. the muscular rings in Mean resting pressure = 14the middle and upper Maximum resting pressure = 20portion of anal canal Mean squeeze pressure = 27

Maximum squeeze pressure = 51E Unreported significative 16 Combined ruptures of EAS Absent Hypotonia at resting pressure, female data and IAS localized in low pressure increasing age 74 the anterior area of in squeeze.

the muscular rings in Mean resting pressure =30 the middle and lower Maximum resting pressure = 35portion of anal canal Mean squeeze pressure = 39

Maximum squeeze pressure = 49

External anal sphincter (EAS), internal anal sphincter (IAS)

2. Processing of adipose tissue with Lipogems®

deviceThe aspirated fat was immediately processed inthe Lipogems® closed system (Figure 1). Thisdisposable device progressively reduces the sizeof the fragments of adipose tissue from spher-oidal cluster with a diameter from 2.5 to 3.5 mmto a micro-cluster from 400 to 900 µm, whileeliminating oily substances and blood residueswhich have pro-inflammatory properties. Themechanical processes of microfracturing, wash-ing and filtration take place in a system com-pletely immersed in physiological solutionavoiding the presence of air, in order to makepossible the reduction in volume and minimizeany traumatic action on cellular products. Thesystem allows harvesting Lipogems® microfrac-tured adipose tissue rich in pericytes and MSCswhich are naturally present in adipose tissueniche37-39,58. The purified Lipogems fat tissue wascollected in a 60 cc syringe and positioned togently decant by gravity in order to remove ex-cessive saline solution. Then the product of li-pogems tissue is transferred in several 1 ccsyringes to be re-injected in the same patient.

3. Re-inoculation in the patient On endoanal ultrasound guidance, internal andexternal anal sphincter were identified (Figure2). Using a special disposable 19G cannula withblunt tip, Lipogems® tissue was inoculated in themuscle defects (Figure 2a), in the intersphinteric

space (Figure 2b) and all around circularly in theremaining portions of the external anal sphinc-ter (Figure 2c). Luer lock syringes ranging from1 ml are used to inject the ideal amount of tissuein each treatment. In patient B, C, and E, whereit was evident a various degrees of deficiency in-nervation of the sphincter muscles, Lipogems®

was also injected along the course of the puden-dal nerve with a transperineal technique ultra-sound-guided59. The fat micrograft was injectedin multiple tunnels into the tissues taking care todeposit it in small quantities (1 ml) in only oneplace each time, from distal to proximal.After the procedure, postoperative pain was eval-uated by compilation of a Visual Numeric Scale(VNS, score 0-10) by the patients. All patientswere re-investigated at 3, 6, 12, 18 and 24months from the treatment to revaluate the phys-iological contractility of the anal sphincters, thesymptoms of fecal incontinence, the quality oflife and the sonographic features in the site of in-oculation of the Lipogems® by repeating thesame preoperative examinations. The surgicalprocedures were performed by A.G. and C.T. Allthe diagnostic examinations, preoperative andpostoperative were performed by A.G. and byanother independent investigator R.V.

STATISTICAL ANALYSISThe results are expressed as mean ± SD (standarddeviation). The statistical significance was deter-mined by the two-tailed Student’s t test. Differenceswere considered statistically significant at p<0.05.

A. Giori, C. Tremolada, R. Vailati, S.E. Navone, G. Marfia, A.I. Caplan4

Figure 1. Lipogems device.Lipogems device is a com-plete closed system filledwith physiologic solution. Itallows the reduction of thesize of lipoaspirate clustersafter washing of oil, bloodand cellular debris. A, Sacwith physiologic solution; B,Syringe with lipoaspirateclusters; C, Washing cham-ber containing marbles forthe emulsion of fluid andelimination of oil and bloodagainst gravity; D, Mechani-cal filters; E, Syringe withclusters of reduced size; F,Sac with waste oil and blood.

RESULTSSURGICAL PROCEDUREIn all 5 patients, the procedure was successfullycompleted without intraoperative or postoperativecomplications. The median operation time, includ-ing the three different phases of harvesting, pro-cessing and reintroduction of the substance, was 73minutes with most time used for reinjections. An av-erage of 330 cc of lipoaspirate was collected fromeach patients. As a result of processing with thetechnique Lipogems®, the average amount of prod-uct obtained, ready for injection in patients, was 90cc. During procedures, no problem of abnormal ac-cumulation of Lipogems®, nor needle clogging, oc-curred.

QUALITY OF LIFEPatients have well tolerated the procedure and the me-dian postoperative pain evaluated by VNS (score 0-10) was 1.4, mainly referred to the site of abdominalfat suction. The postoperative hospitalization was 1day in all patients. Patients’ satisfaction for the treat-ment was very good and the median Fecal Inconti-nence Quality of Life (FIQL) score for all patients hasincreased from preoperative values. After 3 monthsfrom treatment, lifestyle improved from 1.8 to 3.4,coping from 1.6 to 3.6, depression from 2.1 to 3.4 andembarrassment from 1.9 to 3.5, remaining quite stablefor the 2 years of follow up as shown in Figure 3.

In the 5 patients we reported a significant im-provement in the average values of the overallWexner Incontinence Score that has changed from apreoperative value of 14.0 to value of 3.4 at assess-ment three months after treatment, to value of 4.0 at6 months and to value of 4.4 at one year. During thesecond year of follow up values remained stable witha moderate improvement trend, recording values of4.2 at 18 months and 4.2 at 24 months (Figure 4).Moreover, the comparison of Wexner IncontinenceScore measured pre-implantation and at 3, 6, 12, 18,24 months in all 5 patients indicates a significant re-duction in the Wexner score that decrease by a meanof 14.0 units to 3.4 units at 3 months and remainedstable over the time course of our study (Figure 5).

MANOMETRIC FINDINGSGlobal improvement was also reported by manomet-ric findings. After treatment, the overall mean valuesof the anal pressure at rest and in squeeze recorded byanorectal manometry was improved in all the five pa-tients as reported in the graph in Figure 6. Indeed, the

Lipogems treatment in anal incontinence 5

Figure 2. Lipogems injection. Areas of inoculation of Li-pogems (endoanal ultrasound image at mid anal canal level). a,defect of external and internal anal sphincter; b, intersphintericspace; c, peripheral zone of the external sphincter.

Figure 3. Fecal Incon-tinence Quality of LifeScore. Changes inFecal IncontinenceQuality of Life score in5 patients before and 3,6, 12, 24 months afterLipogems treatment.

mean value at rest of 24 mmHg and in squeeze of 39mmHg reported before regenerative therapy, in-creased respectively to 34 mmHg and 80 mmHg at 3months, to 38 mmHg and 90 mmHg at 6 months, to35 mmHg and 96 mmHg at 12 months, to 52 mmHgand 117 mmHg at 18 months and to 45 mmHg and112 mmHg at 24 months.

Similar results were given for maximum restingand squeeze pressures that increased, respectively,from a median of 38 and 72 mmHg at baseline to 58and 112 mmHg at 3 months, to 56 and 120 mmHg at6 months, to 48 and 137 mmHg at 12 months, to 64and 172 mmHg at 18 months and to 63 and 170mmHg at 24 months after the procedure (Figure 6).

Interesting, a clear increase in the anal contrac-tion pressure was recorded for all the patients 12months after the treatment. Indeed, the contract abil-ity of the sphincters increase significantly after Li-pogems® treatment (Figure 7).

PHYSICAL EXAMINATION3 months after treatment the sphincters relaxationpreviously detectable in all patients was no longerappreciable replaced by an effect of filling in theanal canal whose walls appeared soft and elastic atfinger palpation. From 6 to 12 months was clinicallyevident a reduction of filling in the anal canal whichwas maintained up to 24 months. At the same time

Figure 4. Wexner In-continence Score. Av-erage values of theWexner IncontinenceScore (0-20) for all theenrolled patients beforeand after Lipogemstreatment. *p<0.05.

Figure 5. Trend of the Wexner IncontinenceScore. Trend of the Wexner Incontinence Score(0-20) for each patient before and at 3, 6, 12, 18,24 months after Lipogems treatment. * p<0.05.

6

from 6 months up to 24 months was clearly ob-served at palpation a progressive increase in thecontractile activity of the anal sphincters. Further-more, after 3 months it was appreciated in patient Cand E the presence of a moderate anocutaneous re-flex not present prior to the treatment, while in pa-tient B was evidenced a more active reflex.

ULTRASOUND FINDINGSDuring intraoperative Lipogems® treatment, re-spect to preopretaive endoanal ultrasonography(Figure 8a), the deposits of Lipogems® were iden-tifiable by the ultrasound scan as hyperechoic spots(Figure 8b).

7

Figure 6. Mean and maximal anal squeezeand resting pressure. Change in mean andmaximal anal squeeze and resting pressurescores in 5 patients before and 3, 6, 12, 18, 24months after Lipogems treatment.

Figure 7. Manometric curves. Patient A:manometric curves in squeeze recorded be-fore and 12 months after Lipogems treat-ment.

Three months after Lipogems® treatment, en-doanal ultrasonography evidenced only an initial re-sorption of the depots of Lipogems with persistence ofwidespread hyperechoic spots at sites of inoculation(Figure 8c). From 6 to 12 months ultrasonography de-tected a progressive resorption of the hyperechoicspots of Lipogems tissue product (Figure 8d). At thesame time, endoanal ultrasound identified increasingof muscle fibers at the site of previous injury and a re-covery of the circle of the anal sphincters still evidentat 24 months when deposits of Lipogems tissue wereno longer evident (Figure 8e, 8f).

DISCUSSION

As a result of preliminary reports of efficacy andsafety in different medical specialties, regenerativetherapy with the use of autologous stem cells,freshly harvested or culture-expanded, recently hasbeen used for treating those conditions that usually

are unmet and result in poor outcomes or invasivesurgery58. In recent years, autologous stem cellshave been used for experimental treatment of pa-tients with urinary incontinence and anal inconti-nence. In these studies, bone marrow and musculartissue constitutes the most frequent source of adultMSCs35,60-62, although this cell population is quiterare in these tissues (0.001%-0.01%)63. Recently,the human adipose tissue has been identified as aconvenient source of MSCs, generically referred toas “adipose-derived” stem cells (ADSCs)64. In thisstudy we report, for the first time, the use of a fattissue regenerative product (Lipogems®) obtainedwith a closed, non-enzymatic method37,39, to treatpatients with anal incontinence. Other in vitro ob-servations indicate that MSCs are encapsulated inthe fat cell preparation and are the active agents ofthe Lipogems® procedure.

Lipogems tissue appears as a fat grafting proce-dure but the fat tissue which is injected, comparedto standard lipoaspirate, is composed of very smallclusters with viable and intact pericytes and MSCsin their own natural niche36,37. The particular high


Figure 8. Endoanal ultrasonography. Endoanalultrasonography images of a representative patient,Patient A, at preoperative, intraoperative stage and at3, 6, 12, 24 months after treatment. A, Preoperativeendoanal ultrasonography*. Lesion of the externaland internal anal sphincter localized in the anteriorarea of the muscular rings; B, Intraoperative ultra-sonography*. Diffuse hyperechoic spots at the sitesof inoculation of Lipogems; C, Ultrasonography* atthe 3rd month. Partial resorption of hyperechoic spotsof Lipogems; D, Ultrasonography* at the 6th month.Appearance of initial development of new musclefibers at the site of the lesions. Are still evident fewhyperechoic spots of Lipogems; E, and F, Ultra-sonography*, respectively, at the 12th and 24th

month. The image show a kind of muscle repair. Nomore evidence of hyperechoic depots of Lipogems*(scanned at mid anal canal level).

concentration of stromal-vascular fraction presentin clusters of Lipogems® tissue, after the elimina-tion of most of the fat cells during the processing inthe device Lipogems®, is also detectable by thecharacteristics of the ultrasound image achievedwith endoanal ultrasonography. In fact, at the ultra-sound scan, the areas of inoculation of Lipogems®

product appear hyperechoic, indicative for highconcentration of hyperdense cells (Figure 8b),whereas, the adipose tissue has a lower densitywhich gives hypoechoic ultrasound images65,66.

The technique of injection into the patients hasproven to minimally traumatic and very smooth dueto the fluid nature of Lipogems® which can easilypass through fine sharp or blunt needles (21 up to 25gouge) and can uniformly be distributed in targettissues. The method of distribution of the mi-crofragmented fat graft that we employed (Figures2, 8), more than obtaining a favorable bulking ef-fect on the anal canal, provides substance able to re-pair lesions of the anal sphincter apparatus related tomuscle and nerve. Moreover, as fecal incontinencecauses considerable apprehension, we used a vali-dated instrument to address personal implicationsin patient quality of life. We demonstrated that afterLipogems® treatment, the FIQL score for all patientsincreased from preoperative condition.

Indeed, regarding the FIQL score described byRockwood on psychometric evaluation, based on fourscale (Lifestyle, Coping and behavior, Depression/self-perception, Embarassment) in patients affected byfecal incontinence and in subjects known not to haveFecal Incontinence, but other gastrointestinal problems(control group), our patients, after 3 months of treat-ment and for all the follow-up period, showed the samevalue (3<FIQL<4) to the control population57.

By a comparison of patients data, in our experi-ence, we observed a slight worsening of symptomsof incontinence simultaneous to a moderate de-crease of resting pressure in the interval between the3rd month and 12th month (Figures 5, 6). In this sameperiod of time, endoanal ultrasound revealed thatthe deposits of inoculated Lipogems®, were gradu-ally reabsorbing, as evident in Figure 8c, 8d. Later,between the 12th and the 24 month, there has been astabilization of the symptoms of incontinence, whilethere was a small increase in pressure at rest asso-ciated with an evident increase in mean squeezepressure and maximum squeeze pressure. Duringthe second year of follow-up, after the absorptionof the cellular component of adipose tissue, the ul-trasound image showed repair of the anal sphincterwith restitutio ad integrum of the muscle fibers (Fig-

ure 8e). The slight worsening of the symptoms ofincontinence between the 3rd and 12th month is, inour opinion, attributed to absorption of the adiposecomponent of Lipogems® with loss of bulking ef-fect which usually occurs after 6 months to 1 year asis also observed with treatments employing the tra-ditional technique of lipofilling which always re-quire multiple filling41,15,67.

An attractive feature was the clear increase in thecontractile ability of the sphincters recorded byanorectal manometry after 12 months (Figures 6, 7)when deposits of material inoculated disappeared andultrasound images showed muscle repair. Frudingeret al35 reported the use of autologous myoblast cellscultured from a biopsy of pectoralis muscle injectedinto anal sphincter defect to treat patients with analincontinence due to obstetric injury. They report onlya transitory increasing of the maximum anal squeezepressure at the 1 and 6 month assessments, returnedto baseline at 12 months after treatment. WhileFrudinger et al have used muscle-derived cells pre-pared by enzymatic/chemical procedure and culti-vated, we used a tissue graft prepared mechanicallyand in the same surgical session.

The Lipogems® tissue graft preserves the tissuemicro-structure with intact niche that allows a func-tional “regenerative unit”. In this way, the MSCsand pericytes contained in the niche are probablyboth more available and functionally intact than iso-lated injected cells to promote tissue regeneration58.In our initial experience, it is possible that the acti-vation of MSCs has produced muscle repair? Thissuggestion is supported by the impressions of otherauthors68 and there are also studies in vivo and invitro on the capacity of the MSCs to produce re-generation of musculoskeletal cells. In a recentstudy, Lorenzi31 described treatment of anal sphinc-ter injury artificially induced in rats with injection ofBM-MSCs. In addition to the functional results, heevaluated histological examinations performed onanimals sacrificed. At the site of injury, he describedan area rich in irregularly placed muscle cells of dif-ferent sizes that indicated the presence of selectiveregeneration of anal sphincters. In a recently article,Kajbafzadeh69 has investigated the inoculation ofautologous muscle progenitor cells in rabbits previ-ously submitted to external anal sphincterotomy. Inaddition to an enhanced anal contractility evident tomanometry, the histomorphological studies of dam-aged external anal sphincter detected a significantdecrease in interstitial fibrosis associated to regen-


erating muscle fibers with a variable orientation,significant for new muscle fiber regeneration.

The improvement in contractility and anal incon-tinence symptoms recorded in patient B and E, butespecially in patient C, are striking with regard tothe improvement of peripheral nerve function. Themechanism for this beneficial effect is not clear andseveral kinds of mechanisms, such as stimulatingneovascularization, immunomodulation, and neu-rotrophic influences, may be hypothesized. How-ever, in the literature there are only experimentalstudies on the regeneration of peripheral nerves withuse of MSCs and there is no sure clinical evidence ofperipheral nerve regrowth after regenerative MSCstherapy25-28,70-74. This single observation is certainlyvery suggestive, but it is to be verified with furtherand more detailed investigations.

CONCLUSIONS

This new technique seems to give remarkable clin-ical results with a very simple regenerative proce-dure and minimally traumatic surgery which is verywell tolerated by patients.

DECLARATION OF INTERESTSCarlo Tremolada is the inventor of intellectual prop-erty used in this study and an equity owner in Li-pogems International, SpA, licensee of the patentsused in this study.

REFERENCES1. Macmillan AK, Merrie AE, Marshall RJ, Parry BR. The

prevalence of fecal incontinence in community-dwellingadults: a systematic review of the literature. Dis Colon Rec-tum 2004; 47: 1341-1349.

2. Rothbarth J, Bemelman WA, Meijerink WJ, StiggelboutAM, Zwinderman AH, Buyze-Westerweel ME, DelemarreJB. What is the impact of fecal incontinence on quality oflife? Dis Colon Rectum 2001; 44: 67-71.

3. Sultan AH, Kamm MA, Hudson CN, Thomas JM, BartramCI. Anal-sphincter disruption during vaginal delivery. NEngl J Med 1993; 329: 1905-1911.

4. Lindsey I, Jones OM, Smilgin-Humphreys MM, Cunning-ham C, Mortensen NJ. Patterns of fecal incontinence afteranal surgery. Dis Colon Rectum 2004; 47:1643-1649.

5. Oberwalder M, Dinnewitzer A, Baig MK, Thaler K, Cot-man K, Nogueras JJ, Weiss EG, Efron J, Vernava AM 3rd,Wexner SD. The association between late-onset fecal in-continence and obstetric anal sphincter defects. Arch Surg2004; 139: 429-432.

6. Vaizey CJ, Norton C, Thornton MJ, Nicholls RJ, KammMA. Long-term results of repeat anterior anal sphincter re-pair. Dis Colon Rectum 2004; 47: 858-863.

7. Zorcolo L, Covotta L, Bartolo DC. Outcome of anteriorsphincter repair for obstetric injury: comparison of earlyand late results. Dis Colon Rectum 2005; 48: 524-531.

8. Baeten CG, Bailey HR, Bakka A, Belliveau P, Berg E, BuieWD, Burnstein MJ, Christiansen J, Coller JA, Galandiuk S,LaFontaine LJ, Lange J, Madoff RD, Matzel KE, PåhlmanL, Parc R, Reilly JC, Seccia M, Thorson AG, Vernava AM3rd, Wexner S. Safety and efficacy of dynamic graciloplastyfor fecal incontinence: report of a prospective, multicentertrial. Dynamic Graciloplasty Therapy Study Group. DisColon Rectum 2000; 43: 743-751.

9. Lehur PA, Glemain P, Bruley des Varannes S, Buzelin JM,Leborgne J. Outcome of patients with an implanted artifi-cial anal sphincter for severe faecal incontinence: a singleinstitution report. Int J Colorectal Dis 1998; 13: 88-92.

10. Norderval S, Oian P, Revhaug A, Vonen B. Anal inconti-nence after obstetric sphincter tears: outcome of anatomicprimary repairs. Dis Colon Rectum 2005; 48: 1055-1061.

11. Vonthein R, Heimerl T, Schwandner T, Ziegler A. Electri-cal stimulation and biofeedback for the treatment of fecalincontinence: a systematic review. Int J Colorectal Dis2013; 28: 1567-1577.

12. Lim JT, Hastie IA, Hiscock RJ, Shedda SM. Sacral nervestimulation for fecal incontinence: long-term outcomes.Dis Colon Rectum 2011; 54: 969-974.

13. Shafik A. Perianal injection of autologous fat for treatment ofsphincteric incontinence. Dis Colon Rectum 1995; 38: 583-587.

14. Shafik A. Polytetrafluoroethylene injection for the treatmentof partial fecal incontinence. Int Surg 1993; 78: 159-161.

15. Vaizey CJ, Kamm MA. Injectable bulking agents for treat-ing faecal incontinence. Br J Surg 2005; 92: 521-527.

16. Ratto C, Parello A, Donisi L, Litta F, De Simone V, Spaz-zafumo L, Giordano P. Novel bulking agent for faecal in-continence. Br J Surg 2011; 98: 1644-1652.

17. Bernardo ME, Pagliara D, Locatelli F. Mesenchymal stro-mal cell therapy: a revolution in Regenerative Medicine?Bone Marrow Transplant 2012; 47: 164-171.

18. Caplan AI. Adult mesenchymal stem cells for tissue engi-neering versus regenerative medicine. J Cell Physiol 2007;213: 341-347.

19. Caplan AI, Dennis JE. Mesenchymal stem cells as trophicmediators. J Cell Biochem 2006; 98: 1076-1084.

20. Caplan AI, Correa D. The MSC: an injury drugstore. CellStem Cell 2011; 9: 11-15.

21. Herzog EL, Chai L, Krause DS. Plasticity of marrow-de-rived stem cells. Blood 2003; 102: 3483-3493.

22. Muguruma Y, Reyes M, Nakamura Y, Sato T, MatsuzawaH, Miyatake H, Akatsuka A, Itoh J, Yahata T, Ando K, KatoS, Hotta T. In vivo and in vitro differentiation of myocytesfrom human bone marrow-derived multipotent progenitorcells. Exp Hematol 2003; 31: 1323-1330.

23. Santa María L, Rojas CV, Minguell JJ. Signals from dam-aged but not undamaged skeletal muscle induce myogenicdifferentiation of rat bone-marrow-derived mesenchymalstem cells. Exp Cell Res 2004; 300: 418-426.

24. Bossolasco P, Corti S, Strazzer S, Borsotti C, Del Bo R,Fortunato F, Salani S, Quirici N, Bertolini F, Gobbi A,Deliliers GL, Pietro Comi G, Soligo D. Skeletal muscledifferentiation potential of human adult bone marrow cells.Exp Cell Res 2004; 295: 66-78.


25. Park BW, Kang DH, Kang EJ, Byun JH, Lee JS, MaengGH, Rho GJ. Peripheral nerve regeneration using autolo-gous porcine skin-derived mesenchymal stem cells. J Tis-sue Eng Regen Med 2012; 6: 113-124.

26. Matthes SM, Reimers K, Janssen I, Liebsch C, Kocsis JD,Vogt PM, Radtke C. Intravenous transplantation of mes-enchymal stromal cells to enhance peripheral nerve regen-eration. Biomed Res Int 2013; 2013: 573169.

27. Frattini F, Lopes FR, Almeida FM, Rodrigues RF, BoldriniLC, Tomaz MA, Baptista AF, Melo PA, Martinez AM.Mesenchymal stem cells in a polycaprolactone conduit pro-mote sciatic nerve regeneration and sensory neuron sur-vival after nerve injury. Tissue Eng Part A 2012; 18:2030-2039.

28. Pereira Lopes FR, Frattini F, Marques SA, Almeida FM,de Moura Campos LC, Langone F, Lora S, Borojevic R,Martinez AM. Transplantation of bone-marrow-derivedcells into a nerve guide resulted in transdifferentiation intoSchwann cells and effective regeneration of transectedmouse sciatic nerve. Micron 2010; 41: 783-790.

29. Yiou R, Yoo JJ, Atala A. Restoration of functional motorunits in a rat model of sphincter injury by muscle pre-cursor cell autografts. Transplantation 2003; 76: 1053-1060.

30. Yiou R, Dreyfus P, Chopin DK, Abbou CC, Lefaucheur JP.Muscle precursor cell autografting in a murine model ofurethral sphincter injury. BJU Int 2002; 89: 298-302.

31. Lorenzi B, Pessina F, Lorenzoni P, Urbani S, Vernillo R,Sgaragli G, Gerli R, Mazzanti B, Bosi A, Saccardi R,Lorenzi M. Treatment of experimental injury of analsphincters with primary surgical repair and injection ofbone marrow-derived mesenchymal stem cells. Dis ColonRectum 2008; 51: 411-420.

32. Aghaee-Afshar M, Rezazadehkermani M, Asadi A,Malekpour-Afshar R, Shahesmaeili A, Nematollahi-ma-hani SN. Potential of human umbilical cord matrix and rab-bit bone marrow-derived mesenchymal stem cells in repairof surgically incised rabbit external anal sphincter. DisColon Rectum 2009; 52: 1753-1761.

33. Strasser H, Marksteiner R, Margreiter E, Pinggera GM,Mitterberger M, Frauscher F, Ulmer H, Fussenegger M,Kofler K, Bartsch G. Autologous myoblasts and fibroblastsversus collagen for treatment of stress urinary incontinencein women: a randomised controlled trial. Lancet 2007; 369:2179-2186.

34. Kajbafzadeh AM, Elmi A, Payabvash S, Salmasi AH,Saeedi P, Mohamadkhani A, Sadeghi Z, Nikfarjam L.Transurethral autologous myoblast injection for treatmentof urinary incontinence in children with classic bladderexstrophy. J Urol 2008; 180: 1098-1105.

35. Frudinger A, Kölle D, Schwaiger W, Pfeifer J, Paede J,Halligan S. Muscle-derived cell injection to treat anal in-continence due to obstetric trauma: pilot study with 1 yearfollow-up. Gut 2010; 59: 55-61.

36. Tremolada C, G Palmieri and C Ricordi. Adipocyte trans-plantation and stem cells: plastic surgery meets regenera-tive medicine. Cell Transplant 2010; 19: 1217-1223.

37. Bianchi F, Maioli M, Leonardi E, Olivi E, Pasquinelli G,Valente S, Mendez AJ, Ricordi C, Raffaini M, TremoladaC, Ventura C. A new nonenzymatic method and device toobtain a fat tissue derivative highly enriched in pericyte-like elements by mild mechanical forces from humanlipoaspirates. Cell Transplant 2013; 22: 2063-2077.

38. Maioli M, Rinaldi S, Santaniello S, Castagna A, PigliaruG, Delitala A, et al. Radio electric asymmetric conveyedfields and human adipose-derived stem cells obtained witha non-enzymatic method and device: a novel approach tomultipotency. Cell Transplant 2013; 22: 1227-1235.

39. Carelli S, Messaggio F, Canazza A, Hebda DM, Caremoli F,Latorre E, Grimoldi MG, Colli M, Bulfamante G, Tremo-lada C, Giulio AM, Gorio A. Characteristics and Propertiesof Mesenchymal Stem Cells Derived from Micro-frag-mented Adipose Tissue. Cell Transplant 2014 May 6.

40. Coleman SR. Facial recontouring with lipostructure. ClinPlast Surg 1997; 24: 347-350.

41. Bernardi C, Favetta U, Pescatori M. Autologous fat injec-tion for treatment of fecal incontinence: manometric andechographic assessment. Plast Reconstr Surg 1998; 102:1626-1628.

42. Nordström RE, Wang J, Fan J. “Spaghetti” fat grafting: anew technique. Plast Reconstr Surg 1997; 99: 917-918.

43. Riazi AM, Kwon SY, Stanford WL. Stem cell sources forregenerative medicine. Methods Mol Biol 2009; 482: 55-90.

44. Daley GQ, Scadden DT. Prospects for stem cell-based ther-apy. Cell 2008; 132: 544-548.

45. Baglioni S, Francalanci M, Squecco R, Lombardi A, Can-tini G, Angeli R, Gelmini S, Guasti D, Benvenuti S, An-nunziato F, Bani D, Liotta F, Francini F, Perigli G, SerioM, Luconi M. Characterization of human adult stem-cellpopulations isolated from visceral and subcutaneous adi-pose tissue. FASEB J 2009; 23: 3494-3505.

46. Fraser JK, Wulur I, Alfonso Z, Hedrick MH. Fat tissue: anunderappreciated source of stem cells for biotechnology.Trends Biotechnol 2006; 24: 150-154.

47. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Dou-glas R, Mosca JD, Moorman MA, Simonetti DW, Craig S,Marshak DR. Multilineage potential of adult human mes-enchymal stem cells. Science 1999; 284: 143-147.

48. Mitchell JB, McIntosh K, Zvonic S, Garrett S, Floyd ZE,Kloster A, i Halvorsen Y, Storms RW, Goh B, Kilroy G, WuX, Gimble JM. Immunophenotype of human adipose de-rived cells: temporal changes in stromal-associated and stemcell-associated markers. Stem Cells 2006; 24: 376-385.

49. DeUgarte DA, Alfonso Z, Zuk PA, Elbarbary A, Zhu M,Ashjian P, Benhaim P, Hedrick MH, Fraser JK. Differentialexpression of stem cell mobilization-associated moleculeson multi-lineage cells from adipose tissue and bone mar-row. Immunol Lett 2003; 89: 267-270.

50. Marfia G, Campanella R, Navone S, Zucca I, Scotti A, Fig-ini M, Di Vito C, Alessandri G, Riboni L, Parati E. Poten-tial use of human adipose mesenchymal stromal cells forintervertebral disc regeneration: a preliminary study onbiglycan-deficient murine model of chronic disc degener-ation. Arthritis Res Ther 2014; 16: 457.

51. Murphy MB, Moncivais K, Caplan AI. Mesenchymal stemcells: environmentally responsive therapeutics for regen-erative medicine. Exp Mol Med 2013; 45: e54.

52. Dimarino AM, Caplan AI, Bonfield TL. Mesenchymalstem cells in tissue repair. Front Immunol 2013; 4: 201-210.

53. Somoza RA, Welter JF, Correa D, Caplan AI. Chondro-genic differentiation of mesenchymal stem cells: chal-lenges and unfulfilled expectations. Tissue Eng Part B Rev2014; 20: 596-608.


54. Kean TJ, Lin P, Caplan AI, Dennis JE. MSCs: DeliveryRoutes and Engraftment, Cell-Targeting Strategies, and Im-mune Modulation. Stem Cells Int 2013; 2013: 73274.

55. Traktuev DO, Prater DN, Merfeld-Clauss S, SanjeevaiahAR, Saadatzadeh MR, Murphy M, Johnstone BH, IngramDA, March KL. Robust functional vascular network for-mation in vivo by cooperation of adipose progenitor andendothelial cells. Circ Res 2009; 104: 1410-1420.

56. Jorge JM, Wexner SD. Etiology and management of fecalincontinence. Dis Colon Rectum 1993; 36: 77-97.

57. Rockwood TH, Church JM, Fleshman JW, Kane RL,Mavrantonis C, Thorson AG, Wexner SD, Bliss D, LowryAC. Fecal incontinence quality of life scale: Quality of lifeinstrument for patients with fecal incontinence. Dis ColonRectum 2000; 43: 9-17.

58. Murphy MB, Moncivais K, Caplan AI. Mesenchymal stemcells: environmentally responsive therapeutics for regen-erative medicine. Exp Mol Med 2013; 45: e54.

59. Gruber H, Kovacs P, Piegger J, Brenner E. New, simple,ultrasound-guided infiltration of the pudendal nerve: topo-graphic basics. Dis Colon Rectum 2001; 44: 1376-1380.

60. Strasser H, Marksteiner R, Margreiter E, Pinggera GM, Mitter-berger M, Fritsch H, Klima G, Rädler C, Stadlbauer KH,Fussenegger M, Hering S, Bartsch G. Stem cell therapy for uri-nary incontinence. Urologe A 2004; 43: 1237-1241.

61. Furuta A, Jankowski RJ, Honda M, Pruchnic R, YoshimuraN, Chancellor MB. State of the art of where we are at usingstem cells for stress urinary incontinence. Neurol Urodyn2007; 26: 966-971.

62. Smaldone MC, Chancellor MB. Muscle derived stem celltherapy for stress urinary incontinence. World J Urol 2008;26: 327-332.

63. Nishida S, Endo N, Yamagiwa H, Tanizawa T, TakahashiHE. Number of osteoprogenitor cells in human bone mar-row markedly decreases after skeletal maturation. J BoneMiner Metab 1999; 17: 171-177.

64. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ,Benhaim P, Lorenz HP, Hedrick MH. Multilineage cellsfrom human adipose tissue: implications for cell-basedtherapies. Tissue Eng 2001; 7: 211-228.

65. Costantini M, Cipriani A, Belli P, Bufi E, Fubelli R, Vis-conti G, Salgarello M, Bonomo L. Radiological findings in

mammary autologous fat injections: a multi-technique eval-uation. Clin Radiol 2013; 68: 27-33.

66. Pienaar WE, McWilliams S, Wilding LJ, Perera IT. The im-aging features of MACROLANE™ in breast augmenta-tion. Clin Radiol 2011; 66: 977-983.

67. Maeda Y, Laurberg S, Norton C. Perianal injectable bulk-ing agents as treatment for faecal incontinence in adults.Cochrane Database Syst Rev 2010; (5): CD007959.

68. Aghaee-Afshar M, Rezazadehkermani M, Asadi A,Malekpour-Afshar R, Shahesmaeili A, Nematollahi-ma-hani SN. Potential of human umbilical cord matrix and rab-bit bone marrow-derived mesenchymal stem cells in repairof surgically incised rabbit external anal sphincter. DisColon Rectum 2009; 52: 1753-1761.

69. Kajbafzadeh AM, Elmi A, Talab SS, Esfahani SA,Tourchi A. Functional external anal sphincter reconstruc-tion for treatment of anal incontinence using muscle pro-genitor cell auto grafting. Dis Colon Rectum 2010; 53:1415-1421.

70. Park BW, Kang DH, Kang EJ, Byun JH, Lee JS, MaengGH, Rho GJ. Peripheral nerve regeneration using autolo-gous porcine skin-derived mesenchymal stem cells. J Tis-sue Eng Regen Med 2012; 6: 113-124.

71. Hermann A, List C, Habisch HJ, Vukicevic V, Ehrhart-Bornstein M, Brenner R, Bernstein P, Fickert S, Storch A.Age-dependent neuroectodermal differentiation capacityof human mesenchymal stromal cells: limitations for au-tologous cell replacement strategies. Cytotherapy 2010; 12:17-30.

72. Levy YS, Bahat-Stroomza M, Barzilay R, Burshtein A,Bulvik S, Barhum Y, Panet H, Melamed E, Offen D. Re-generative effect of neural-induced human mesenchymalstromal cells in rat models of Parkinson’s disease. Cy-totherapy 2008; 10: 340-352.

73. Pereira Lopes FR, Frattini F, Marques SA, Almeida FM,de Moura Campos LC, Langone F, Lora S, Borojevic R,Martinez AM. Transplantation of bone-marrow-derivedcells into a nerve guide resulted in transdifferentiation intoSchwann cells and effective regeneration of transectedmouse sciatic nerve. Micron 2010; 41: 783-790.

74. Di Summa PG, Kingham PJ, Raffoul W, Wiberg M,Terenghi G, Kalbermatten DF. Adipose-derived stem cellsenhance peripheral nerve regeneration. J Plast ReconstrAesthet Surg 2010; 63: 1544-1552.


recovery of function in anal incontinence after micro ... · anal incontinence is common and...

Documents