studies in flexor tendon reconstruction: biomolecular modulation of tendon repair and tissue...

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2011WEILANDMEDAL RECIPIENT

Studies in Flexor Tendon Reconstruction: Biomolecular

Modulation of Tendon Repair and Tissue Engineering

James Chang, MD

The Andrew J. Weiland Medal is presented each year by the American Society for Surgeryof the Hand and the American Foundation for Surgery of the Hand for a body of work relatedto hand surgery research. This essay, awarded the Weiland Medal in 2011, focuses on theclinical need for flexor tendon reconstruction and on investigations into flexor tendonbiology. Reconstruction of the upper extremity is limited by 2 major problems after injuryor degeneration of the flexor tendons. First, adhesions formed after flexor tendon repair cancause decreased postoperative range of motion and hand function. Second, tendon losses canresult from trauma and degenerative diseases, necessitating additional tendon graft material.Tendon adhesions are even more prevalent after tendon grafting; therefore these 2 problemsare interrelated and lead to considerable disability. The total costs in terms of disability andinability to return to work are enormous. In this essay, published work from the past 12 yearsin our basic science laboratory is summarized and presented with the common theme of usingmolecular techniques to understand the cellular process of flexor tendon wound healing andto create substances and materials to improve tendon repair and regeneration. These areefforts to address 2 interrelated and clinically relevant problems that all hand surgeons facein their practice. (J Hand Surg 2012;37A:552–561. Copyright © 2012 by the AmericanSociety for Surgery of the Hand. All rights reserved.)

Key words Decellularization, flexor tendon, growth factors, tissue engineering.

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IDENTIFICATION OF TRANSFORMING GROWTHFACTOR (TGF)-�1 AND OTHER GROWTHACTORS IN FLEXOR TENDON REPAIR

lexor tendon adhesion formation remains a basic, un-olved problem in hand surgery. Even with improvedepair techniques and early active motion therapy, re-

From the Departments of Surgery (Plastic Surgery) and Orthopedic Surgery, Robert A. Chase HandCenter, Stanford University Medical Center, Palo Alto, CA.

Received for publication December 2, 2011; accepted in revised form December 12, 2011.

The research described herein was funded by several Federal VA Merit Review and ASSH/AFSHgrants. All animal protocols were approved by our Institutional Animal Care and Use Committee(IACUC),andallexperimentswereperformedinanAssociationforAssessmentandAccreditationofLaboratory Animal Care (AAALAC) accredited animal facility.

No benefits in any form have been received or will be received related directly or indirectly to thesubject of this article.

Corresponding author: James Chang, MD, Division of Plastic and Reconstructive Surgery, Stan-ford University Medical Center, 770 Welch Road, Suite 400, Palo Alto, CA 94304; e-mail:[email protected].

0363-5023/12/37A03-0024$36.00/0

ddoi:10.1016/j.jhsa.2011.12.028

552 � © ASSH � Published by Elsevier, Inc. All rights reserved.

ults are limited by adhesions within the tight fibro-sseous sheath in zone II.1

Previous work over many years has focused on con-rolling flexor tendon scar formation biochemically, butuch laboratory work has not led to effective clinicalpplications.2–6 Synthetic materials such as nylon and

polytetrafluoroethylene used to create artificial sheathshave not proved successful.7 In addition, agents such asntihistamines, steroids, dimethyl sulfoxide, and hyal-ronic acid have not markedly decreased the amount ofostoperative adhesions.8–11

In the past decade, wound healing research has led tohe characterization of growth factors and their role inissue repair.12,13 Several growth factors have beenound to have roles in tendon wound healing.14,15 Au-

thors who have pioneered this field in hand surgeryhave included Thomopoulos et al,16 Tsubone et al,17

and Wang et al.18

In our first work in the field, we examined thepregulation of TGF-�1 in a rabbit zone II flexor ten-
on wound-healing model.19 We performed in situ hy-
mailto:[email protected]

STUDIES IN FLEXOR TENDON RECONSTRUCTION 553

bridization on frozen sections of the tendon repair spec-imens using probes specific for TGF-�1 mRNA. Asmall number of tenocytes exhibited expression ofTGF-�1 mRNA at baseline in unwounded control ten-don specimens. The surrounding tendon sheath in thesecontrol specimens also revealed low numbers of fibro-blasts and inflammatory cells expressing TGF-�1mRNA. In contrast, flexor tendons subjected to transec-tion and repair exhibited notably elevated TGF-�1mRNA in both resident tenocytes and infiltrating fibro-blasts and inflammatory cells from the tendon.

These data demonstrated that (1) normal unwoundedtenocytes and tendon sheath cells were capable ofTGF-�1 production, (2) this growth factor was elevatedin the tendon wound environment as evidenced bymRNA upregulation, and (3) this upregulation ofTGF-�1 in both intrinsic tenocytes and extrinsic tendonsheath fibroblasts and inflammatory cells supporteddual mechanisms for tendon repair.

After this initial work localizing TGF-�1, we per-formed similar experiments to identify other growthfactors and cytokines, including platelet-derived growthfactor, interleukin-2, and basic fibroblast growth factor(bFGF).20 Basic fibroblast growth factor is a 146–amino acid polypeptide known to be a potent stimulatorof angiogenesis. One of the early events of woundhealing is angiogenesis, in which neovascularizationpromotes delivery of inflammatory cells and fibroblaststo the wound site. In flexor tendon wound healingspecifically, the recruitment of wound repair cells mayinvolve tendon sheath synovial diffusion, migration viaintact vincula, and neovascularization of the tendonsheath. Therefore, we hypothesized that bFGF wouldbe upregulated during flexor tendon wound healing.

In the experiments for bFGF, we found that fewtenocytes and tendon sheath cells expressed bFGFmRNA in unwounded tendons. In contrast, tendonssubjected to transection and repair exhibited increasedsignal for bFGF mRNA in both resident tenocytesalong the epitenon and infiltrating fibroblasts and in-flammatory cells from the tendon sheath. The upregu-lation of bFGF in both intrinsic tenocytes and tendonsheath cells demonstrated that flexor tendon woundhealing involved several cell lines and mechanisms thatmay be redundant.

The TGF-� family binds to 3 distinct classes ofreceptors termed RI, RII, and RIII. In a third study, weanalyzed the temporal and spatial distribution of TGF-�receptor isoforms.21 Immunohistochemical staining ofthe transected and repaired tendons demonstrated up-regulation of TGF-� RI, RII, and RIII protein levels.
Transforming growth factor-� receptor production in
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the experimental group (transection and repair) wasconcentrated in the epitenon and along the repair site.Furthermore, the TGF-� receptor expression levelspeaked at day 14 and decreased by day 56 postopera-tively. In contrast, we observed minimal receptor ex-pression in the untransected and unrepaired control ten-dons. The upregulation of TGF-� receptors duringflexor tendon wound healing provided new targets forbiomolecular modulation of postoperative scar forma-tion.

In these 3 in situ studies, we documented that growthfactors including TGF-� and their corresponding recep-tors were upregulated after flexor tendon injury andrepair. Therefore, they may have a role in flexor tendonwound healing and in the adhesion formation that isobserved clinically. Furthermore, this upregulation oc-curred in 2 distinct areas of the wound: the overlyingtendon sheath and the tendon itself. Because the tendonsheath typically scars to the tendon and restricts tendongliding, strategies that would specifically block TGF-�overexpression in the sheath may block excessive scar-ring without weakening the tendon repair.

The goal would be to manipulate the quantity andbioavailability of these growth factors at surgery toinduce a favorable healing response and to reduce scar-ring. Transforming growth factor-� has numerous bio-logical activities related to wound healing, includingfibroblast and macrophage recruitment, stimulation ofcollagen production, downregulation of proteinase ac-tivity, and increase in metalloproteinase inhibitor activ-ity.22 Three mammalian isoforms of TGF-� exist:TGF-�1, 2, and 3. All 3 isoforms are produced by mostcells active in wound healing, and platelets are majorcontributors.

Transforming growth factor-� has been found toaccelerate the wound-healing process in several mod-els.23 However, this effect may ultimately proceed toofar and result in the pathogenesis of fibrosis, with ex-cessive disordered collagen deposition in dermal tis-sue.24–26 Roberts et al27 documented increased fibrosisin mice after addition of TGF-� in both in vitro and invivo models.

To control scar formation, attention has been di-rected to blocking the actions of TGF-�. Several strat-egies have been used to neutralize TGF-�. Shah etal28,29 were able to control scarring in rat dermalwounds using a neutralizing antibody to TGF-�. Morerecently, TGF-� soluble receptors, which can alsoblock signal transduction, have been successfully iso-lated and used in models of hepatic and pulmonaryfibrosis.30–33 Finally, the neutralization of TGF-� with
natural inhibitors such as decorin and mannose-6-
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554 STUDIES IN FLEXOR TENDON RECONSTRUCTION

phosphate has resulted in decreased fibrosis in modelsof dermal wound healing and glomerulonephritis.34

Mannose-6-phosphate is a simple carbohydrate that isalso known to inhibit TGF-�.35 These natural inhibitorshave structural similarity to betaglycan (TGF-� type IIIreceptor) and can competitively bind TGF-�.

Although promising work has been accomplished inlimiting scar formation in diverse tissues and diseasessuch as dermal wounds, glomerulonephritis, and he-patic fibrosis, less attention has been directed towardblocking TGF-� in flexor tendon wound healing.

IN VITRO STUDIES OF TGF-� IN TENDONCELLSOnce we documented the upregulation of TGF-� andTGF-� receptors in flexor tendon wound healing, wedirected attention toward developing an in vitro modelfor separating the component cells involved. Specifi-cally, we cultured 3 cell lines of collagen-producingcells: tendon sheath fibroblasts (S), epitenon tenocytes(E), and intrinsic tenocytes (T). We isolated these 3 celltypes by microdissection and enzymatic treatment andsubjected them to in vitro stresses. We observed result-ing changes in gene expression.

Using this rabbit model, we examined the effects ofTGF-�1, 2, and 3 on tendon cell proliferation andcollagen production using immunohistochemistry andenzyme-linked immunosorbent assay.36 The addition ofall 3 TGF-� isoforms to cell cultures resulted in asignificant increase in collagen I and III production(P � .05). These increases in collagen production re-sulting from each TGF-� isoform occurred in a dose-dependent manner.

From this study, we concluded that addition ofTGF-� in vitro in tendon cells could result in quantifi-able changes in collagen production. We used collagenproduction as a marker for fibrosis. We then used this invitro model to test and screen anti-scarring agents be-fore in vivo testing in the rabbit model.

The importance of lactate in stimulating the produc-tion of scar has been well described.37,38 Lactate is anearly mediator in the wound-healing process. Tissuehypoxia stimulates lactate production from tissue mac-rophages; lactate, in turn, is a strong stimulant of col-lagen production. We then asked whether lactate couldmimic conditions found early in tendon wound healing.In addition, we investigated whether lactate could stim-ulate TGF-� and collagen production.

In this next study, we examined the effects of lactateon cell proliferation and collagen production in flexortendon cells.39 We isolated 3 separate cell lines—S, E,
and T—from rabbit flexor tendons and cultured them
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separately. With the addition of 25 mmol/L lactate tocell culture media, there was a large increase in collagenproduction by all cell lines. We observed the greatestincreases in the extrinsic tendon sheath cell line. How-ever, the absolute number of cells did not increase withthe addition of lactate. Therefore, lactate increased col-lagen production not by increasing cell proliferation,but by upregulating collagen production by each indi-vidual cell.

This study confirmed that 3 distinct cell lines—sheath, epitenon tenocytes, and endotenon tenocytes—have different potentials for proliferation and extracel-lular matrix production. Extrinsic sheath tenocytes wereconsiderably more proliferative than intrinsic epitenonand endotenon tenocytes. The traditional paradigm ofadhesion formation as either extrinsic or intrinsic wasexpanded to reflect cell proliferation and collagen pro-duction by 3 distinct regions.

In a follow-up study, we investigated the effect oflactate on TGF-� and TGF-� receptor production byflexor tendon cells.40 We also assessed TGF-� func-tional activity by the addition of tendon cell conditionedmedia to mink lung epithelial cells transfected with aluciferase-reporter-gene-expression construct respon-sive to TGF-�. This technique allowed determination ofonly active TGF-� levels.

Supplementation of cell culture media with lactatesignificantly increased all 3 TGF- � peptide and recep-tor isoforms in all 3 cell lines (P � .05). Extrinsictendon sheath fibroblasts exhibited the greatest in-creases in TGF-�1 and TGF-�2 isoforms, whereas en-dotenon tenocytes showed the largest increase for TGF-�3. Epitenon tenocytes exhibited the greatest receptorincreases for receptor isoforms R1 and R2. All 3 tendoncell types showed significant increases in TGF-� func-tional activity when exposed to lactate. Epitenon teno-cytes showed the greatest increase in activity, whereastendon sheath fibroblasts showed the greatest overalllevels of total TGF-� functional activity.

In summary, lactate significantly increased expres-sion of TGF-� peptides (TGF-�1, TGF-�2, and TGF-�3), receptors (R1, R2, and R3), and functional activity,which suggests a common pathway regulating tendoncell collagen production. The extrinsic sheath fibro-blasts had the highest levels of TGF-� production andfunctional activity. Because lactate mimics the earlywound response, lactate upregulation of TGF-� may bean initial event in the tendon wound-healing cascade.41

Extrinsic tendon sheath cells were found to be the mostproliferative component of tendon wound healing.Also, sheath cells were most responsive to lactate and
TGF-� upregulation. Therefore, the sheath, which is
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responsible for adhesion formation, may be targeted.Because specific inhibitors of lactate have not beenavailable, our strategy focused on downstream inhibi-tors of TGF-�.

We performed the first series of in vitro screeningexperiments using antibody to TGF-� as proof of con-cept before screening the natural inhibitors, decorin andM6P. We examined the effectiveness of neutralizingantibody to TGF-�1 (NA-TGF-�1 in blocking TGF-�–induced collagen I production in rabbit flexor tendoncells.42

We supplemented each S, E, and T cell culture with1 ng/mL TGF-� along with increasing doses of NA-TGF-�1 (0.1–2.0 �g/mL). The addition of neutralizingantibody considerably reduced TGF-�–induced colla-gen I production in a dose-dependent manner in all 3cell cultures: S, E, and T. This neutralizing antibodyalso reduced TGF-� functional activity.

This study demonstrated that TGF-� inhibitionthrough its neutralizing antibody was effective incultured flexor tendon cells. Inhibition decreased bothTGF-� functional activity and collagen I production.These results encouraged further experiments to usesuch agents to modulate flexor tendon wound healing inin vivo animal models.

Once we established neutralization of TGF-� byantibodies in vitro, we used this model to test naturalinhibitors. Natural inhibitors have the advantage of be-ing cheaper, less toxic, and less immunogenic thanneutralizing antibodies. The effects of 2 potential natu-ral inhibitors of TGF-�, decorin, and M6P were inves-tigated.43 Both decorin and M6P reduced TGF-�–induced collagen production. In cultured S, E, and Tcells, the stimulatory effects of TGF-� were reduced byboth decorin and M6P. These data supported the use ofdecorin and M6P in in vivo testing. Again, sheath cellswere responsive to downregulation of collagen produc-tion with inhibitors of TGF-�.

IN VIVO MODULATION OF TGF-� IN FLEXORTENDON REPAIRWe performed pilot in vivo studies concurrently withthe above in vitro studies to develop a quantifiablemodel of flexor tendon wound healing and to test theeffect of neutralizing antibody to TGF-�. In the initialstudy, we examined the effect of a neutralizing antibodyto TGF-�1 in a rabbit zone II flexor tendon wound-healing model.44 Adult New Zealand White rabbitsunderwent complete transection of the middle digitflexor digitorum profundus tendon in zone II. We im-mediately repaired the tendons and gave them intraop-
erative infiltration of control phosphate-buffered saline,
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50 �g neutralizing antibody to TGF-�1, or 50 �g eachof neutralizing antibody to TGF-�1 and to TGF-�2.Unoperated rabbits underwent analysis to determine nor-mal proximal interphalangeal (PIP) joint range of motionwith a 125-g load. All experimental rabbits were placedinto casts for 8 weeks to maximize tendon adhesions.

Normal range of motion at the PIP joint using a125-g load on unoperated rabbit forepaws was 93° �6°. Casting of these unoperated forepaws did not reducemotion, with the result of 93° � 4°. Complete transec-tion and repair of the flexor digitorum profundus tendonwith infiltration of control PBS resulted in significantlydecreased PIP joint range of motion: 15° � 6°. How-ever, in the tendon repairs infiltrated with neutralizingantibody to TGF-�1, PIP joint motion increased to32° � 9°. Interestingly, a combination of neutralizingantibody to TGF-�1 and TGF-�2 did not improvepostoperative motion.

Because TGF-�1 is thought to contribute to thepathogenesis of excessive scar formation, the findingssuggested that perioperative biochemical modulation ofTGF-�1 levels could limit flexor tendon adhesion for-mation in vivo. In addition, the rabbit in vivo systemmay be used to quantitate results after application ofother anti–TGF-� agents.

Finally, our laboratory performed in vivo studies totest the efficacy of natural inhibitors of TGF-� in de-creasing postoperative tendon adhesions.45 The goalwas to examine the effectiveness of these 2 naturalinhibitors in blocking TGF-�–induced collagen produc-tion in an in vivo rabbit model of zone II flexor tendonrepair.

We transected and immediately repaired rabbit zoneII flexor tendons. We added a single intraoperative doseof decorin, M6P, or control phosphate-buffered salineto the repair site. Rabbits were killed at 8 weeks post-operatively and forepaws were tested for motion andrepair strength.

Intraoperative application of both low-dose andhigh-dose M6P significantly improved range of motionin operated digits. Both decorin dose-response groupsshowed less improvement in motion. There was nostatistical difference in the mean breaking strength be-tween the control and study groups. Our data suggestedthat a single intraoperative dose of M6P was effective inoptimizing tendon wound healing by improving post-operative range of motion while retaining repairstrength. This simple carbohydrate is ubiquitous, non-immunogenic, and easily produced, which makes it anideal candidate for clinical application.

Further studies are now proposed to determine opti-
mal dosage and delivery method for M6P. In addition,
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human clinical trials for M6P have been ongoing forreduction of scar in dermal wound healing. Once safetyand efficacy are established, the next step would be toconduct human clinical trials using intraoperative M6Pin flexor tendon repair with the hope of allowing im-provement in motion with minimal incremental risk oftendon rupture.

THE NEED FOR ADDITIONAL FLEXOR TENDONGRAFT MATERIALAfter adhesion formation, the second major problem inflexor tendon reconstruction is the need for additionalsources of tendon graft material. The recent wars in Iraqand Afghanistan have unfortunately resulted in manysoldiers returning with mutilating injuries to the upperextremity. The use of autologous tendon grafts hasimproved function in a great number of patients. How-ever, in mutilating hand injuries, severe tendon lossesoutstrip the patient’s own donor tendon supply. In ad-dition, problems remain with adhesions causing limitedexcursion and poor digital function. In a series of ten-don grafts, the procedures resulted in 16% excellent,23% good, 26% fair, and 35% poor results in 43cases.46 These mixed results highlight the problem ofscar formation after tendon grafting.

In the past 20 years, there has been considerableinterest in the use of intrasynovial tendon grafts insteadof the conventional extrasynovial grafts. Intrasynovialgrafts reside inside the tendon sheath and include onlythe flexor digitorum longus tendons of the foot.47 Avail-ability of these tendons is even more limited. Gelber-man’s group48 found that intrasynovial tendons andextrasynovial tendons possess different properties ofhealing, and that grafts of intrasynovial origin moreclosely resemble the cellular properties of the damagedzone II flexor tendon. Histologically, intrasynovial ten-dons differ from extrasynovial tendons by the presenceof a single layer lining of epitenon cells. In vivo caninestudies by Seiler et al49 showed that when these intra-synovial tendons were grafted in the intrasynovialspace, less adhesion formation and better functionresulted. Intrasynovial grafts healed with preserva-tion of the gliding surfaces, whereas the healing ofextrasynovial grafts led to adhesion formation. Theseobservations suggested that extrasynovial tendongrafts functioned primarily as scaffolding for theingrowth of new vessels and cells through the for-mation of adhesions. Despite compelling results fromanimal studies, the technique of intrasynovial tendongrafting is not routinely used clinically because do-nor intrasynovial tendons are limited to the flexor
digitorum longus tendons of the foot.
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Therefore, the next step in tendon reconstructionwould be the production of tissue-engineered intrasy-novial flexor tendon grafts with an intact epitenon celllayer. Compared with other connective tissue typessuch as skin, bone, and cartilage, the ex vivo manufac-turing of flexor tendons has not been studied exten-sively. In addition, most work in tendon tissue engi-neering has focused on reconstruction of extrasynovialtendons and ligaments.

Different scaffold materials have been used for ten-don tissue engineering. Young et al50 showed that fi-broblast-contracted collagen gels could be used to re-construct rabbit patellar tendon defects. Treated tissueshad improved properties including a larger cross-sectional area, better collagen fiber alignment, and bet-ter biomechanical properties at 12 weeks. Others havechosen to use biodegradable synthetic constructs suchas polylactide-co-glycolide for tendon reconstruction.51

The first attempt at tissue engineering of flexor ten-dons specifically was reported in 1994.52 Cao et alisolated calf tenocytes, expanded the cell population,seeded the cultured tenocytes onto polyglycolic acidscaffolds, allowed the constructs to mature in vitro for 1week, and implanted the constructs subcutaneously innude mice. Evaluation revealed gross resemblance ofthe tendon constructs to normal tendons, parallel linearorganization of collagen bundles throughout the con-structs, and comparable tensile strength. However,these attempts at tissue engineering have not led totranslational use in humans owing to limitations in cellseeding and in scaffold strength and biocompatibility.

Our research protocol to create tissue-engineeredintrasynovial flexor tendons uses a strategy of optimiz-ing the scaffold material, the growth conditions, and thecells. Specifically, acellularized flexor tendons are usedas scaffold material, and several proliferative humancell lines are reseeded onto this scaffold. In addition,growth conditions are maximized using biocompatiblegrowth factors and bioreactors.

TISSUE-ENGINEERED FLEXOR TENDON GRAFTSIN A RABBIT MODELOur research group published the use of acellularizedintrasynovial flexor tendons as scaffold material in2009.53 This is a clinically useful scaffold because largequantities of human cadaver flexor tendon can be har-vested, processed, and stored. In addition, it is hypoth-esized that tenocytes will more readily repopulate, andmesenchymal stem cells will more readily differentiate,onto acellularized tendon because this scaffold retainsthe normal collagen architecture of tendon. The use of
acellularized tissue for tissue engineering has been
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well-established for cardiac valves,54,55 peripheralnerve,56 muscle,57 and blood vessels.58

For the past several years, our laboratory used arabbit model for flexor tendon tissue engineering.59,60

We have successfully acellularized tendons, seededthem with various cells, and reimplanted these con-structs into recipient rabbits as tendon grafts. Theseconstructs grossly resembled normal intrasynovial ten-dons, and we found cells both on the surface and in thecore of the construct histologically. These grafts sur-vived long-term in vivo and had comparable strength tonormal tendon grafts.

After we implanted these tissue-engineered tendonconstructs into rabbits, they exhibited a population oftenocyte-like cells; however, we did not know to whatextent these cells were of donor or recipient origin.Furthermore, the temporal distribution was not known.To answer these questions, we extracted tenocytes fromNew Zealand male rabbits, cultured them in vitro, andseeded them onto acellularized rabbit forepaw flexortendons.61 These tendon constructs were then trans-planted to a zone II defect in female recipients. Weexamined tendons after 3, 6, 12, and 30 weeks usingfluorescent in situ hybridization to detect the Y-chro-mosome in the male donor cells.

Donor male cells survived in decreasing numbersover time in the tendon construct until 30 weeks post-transplantation. This study showed that whereas donortenocytes contributed to short-term strength, recipientsite cells migrated into and repopulated the tendonconstruct, thus achieving long-term viability.

Another area of interest was the use of cell and tissuebioreactors to improve both growth of cells in cultureand tendon construct strength. Wang et al62 found thattendon fibroblasts subjected to cyclic mechanicalstretching increased protein expression. Fluid-inducedshear stress also upregulated gene expression.63 Garvinet al64 introduced the concept of mechanical loading ofbioengineered tendon constructs. Using a novel biore-actor system, they were able to create tenocyte-collagengel constructs with tendon-like morphology. Along thisstrategy, we have used bioreactors to precondition bothcells and tendon constructs before reimplantation.

We hypothesized that during postoperative hand mo-bilization, tenocytes experience mechanical shearforces that alter their biology to activate key genesinvolved in tenocyte proliferation and tendon healing. Acell bioreactor capable of producing cyclic strain wasused to provide physiologic conditions to the tenocytesand surrounding cells. The optimal mechanical condi-tions to favor cellular proliferation and collagen pro-
duction, and to maintain morphology in candidate cell
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lines cultured for flexor tendon tissue engineering, wereinvestigated.65 The cell lines tested included primarycultures of epitenon tenocytes (E), sheath fibroblasts(S), bone marrow-derived mesenchymal stem cells, andadipoderived stem cells. Cyclic strain resulted in cellalignment perpendicular to the strain axis, cytoskeletalalignment, and nuclear elongation. These morphologi-cal characteristics were similar to those of normal teno-cytes. These results demonstrated that intermittent cy-clic strain can increase cell proliferation and collagen Iproduction, and maintain tenocyte morphology in vitro.

We then investigated the effect of a tendon tissuebioreactor. In this study, we used a custom bioreactor toapply cyclic mechanical loading onto tissue-engineeredtendon constructs to study ultimate tensile stress, elasticmodulus, construct architecture, and cell orientation.66

Tendon constructs were subjected to a stretch force of1.25 N over a 5-day course. Cyclic bioreactor loadingof tendon constructs increased the ultimate tensile stressand elastic modulus of seeded constructs and altered theconstructs’ collagen architecture and cell orientation.This provided evidence that the tissue bioreactor mayaccelerate the in vitro production of strong, nonimmu-nogenic tendon material.

In addition to bioreactor treatment, we sought tomaximize cell growth using proliferative cell lines andgrowth factors.67 To enhance conditions for the growthof various candidate cell lines, we optimized cell pro-liferation with growth factor supplementation. Platelet-derived growth factor–BB, insulinlike growth factor–1,and bFGF are known to promote tendon healing andtendon cell proliferation. The combination of insulin-like growth factor–1 (100 ng/mL) plus platelet-derivedgrowth factor–BB (50 ng/mL) plus bFGF (5 ng/mL)resulted in the greatest cell proliferation for all 3 tendoncell populations. This combination of insulinlikegrowth factor, platelet-derived growth factor–BB, andbFGF has been used to maximize tenocyte proliferationas well as proliferation of alternative cell populations.

Next, we compared tenocytes with alternative cellpopulations in the rabbit model.68 We investigated 4cell lines: rabbit tenocytes, tendon sheath cells, bonemarrow–derived mesenchymal stem cells, and adi-poderived stem cells (ASC) for use in flexor tendontissue engineering. Specifically, we compared collagenproduction, cell proliferation, cellular senescence, andviability in vitro and in vivo. All 4 cell types weresuccessfully grown in culture and had the characteristicmorphology of fibroblasts, and all stained strongly pos-itive for collagens 1 and 3 and weakly for collagen 2.The 4 groups demonstrated exponential cell growth,
reaching confluence by day 6. All 4 cell types were
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similar and showed almost no senescence even as lateas passage 21. After 6 weeks in vivo, the neotendonshad the characteristic appearance of intrasynovial ten-dons with a single layer of cells on the surface andmultiple cells distributed throughout the core of theneotendon. Therefore, tenocytes, sheath cells, bonemarrow–derived mesenchymal stem cells, and ASCmay all be candidates for tissue engineering. Acellularcollagen scaffolds seeded with all 4 types of cells wereviable in vivo and demonstrated the characteristic ap-pearance of intrasynovial tendons.

The logical succeeding step was to test the tendonbioreactor on tendon constructs seeded with these alter-native cell lines.69 Flexor rear paw rabbit tendons wereacellularized and seeded with ASC or fibroblasts (F)(2 � 106 per construct). The tendon bioreactor appliedcyclic mechanical (1 cycle/min) load of 1.25 N onto thetendon constructs for 5 days. The application of cyclicstrain on seeded tendon constructs improved their bio-mechanical properties, achieving values comparable tofresh tendons. Therefore, the bioreactor was also effec-tive in these alternative cell populations: adipose-derived stem cells and fibroblasts.

The final project in this rabbit model was to implanttissue-engineered tendon grafts into zone II defects toinvestigate the long-term integrity of the neotendons.We produced flexor tendons using all the tissue-engineering steps from the previously described papersand implanted them into zone II defects in rabbit fore-paws. The purpose of this experiment was to evaluatethe in vivo biomechanical strength of explanted con-structs over time.70 Results showed that there were nosignificant differences between experimental groups inultimate tensile strength (UTS) compared with the au-tologous graft control up to 4 weeks. At 10 weeks, therewas a significant decrease in UTS in all groups includ-ing the autologous grafts. At 20 weeks, all groupsshowed an increase in strength compared with 10weeks, and this increase was significant for the tenocytereseeded constructs. At the 20-week time point, all 4groups, including the autologous graft controls, hadregained their UTS compared with the initial 2-weektime point. Our results suggested that flexor tendons canbe tissue engineered successfully and that they maintaintheir biomechanical stability comparable to autologousgrafts in the short term as well as in the long term.

TISSUE-ENGINEERED FLEXOR TENDON GRAFTSIN A HUMAN MODEL: WORK TOWARD ACLINICAL TRIALThe final step in flexor tendon tissue engineering would
be to directly translate this work to hand surgery pa-
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tients with mutilating injuries and severe tendon losses.As with all translational research, the transition from ananimal model to actual patients has major challenges.This required optimization of the protocols for acellu-larization, cell seeding, and bioreactor preconditioningbecause the dimensions of human flexor tendons aredifferent from rabbit flexor tendons in terms of length,width, and density.

We optimized the human flexor tendon acellulariza-tion technique by varying candidate detergents and con-centrations.71 We treated human flexor tendons with0.1% ethylenediaminetetraacetic acid for 4 hours fol-lowed by 24-hour treatments of 1% Triton X-100, 0.1%or 1% tri(n-butyl)phosphate, or 0.1% or 1% sodiumdodecyl sulfate (SDS) in 0.1% ethylenediaminetet-raacetic acid. We reseeded acellularized tendons in asuspension of human dermal fibroblasts to test biocom-patibility. Only SDS treatments significantly decreasedDNA content compared with the DNA content of freshtendons. Histology confirmed these findings. Therewere no decreases in glycosaminoglycans and collagencontent after acellularization with SDS. Importantly,there was no difference in UTS. Reseeded tendonsdemonstrated attachment of viable cells to the tendonsurface using a viability assay and histology. Humanflexor tendons that were acellularized with 0.1% SDS in0.1% ethylenediaminetetraacetic acid for 24 hours hadthe best preservation of biological and mechanicalproperties.

Cell penetration is restricted by the tightly wovenflexor tendon matrix. To further enhance reseeding ofhuman tendons, we evaluated peracetic acid treatmentin optimizing intratendinous cell penetration.72 The op-timal treatment protocol, composed of peracetic acid at5% concentration for 4 hours, produced interfiber gap-ping and increased scaffold porosity, improving cellpenetration and migration. Treated scaffolds did notshow reduced collagen or glycosaminoglycan contentcompared with controls. Treated scaffolds were cyto-toxic neither to attached cells nor to the surrounding cellsuspension. Treated scaffolds also did not show inferiorUTS or extracellular matrix compared with controls.Therefore, peracetic acid treatment of acellularized ten-don scaffolds increased matrix porosity, leading togreater reseeding without biomechanical compromise.

After optimal acellularization and reseeding, it wasimportant to maximize the tissue-engineered con-struct’s biomechanical properties to ensure that the con-struct was in its strongest possible state before reim-plantation.73 Using small- and large-chamber,3-dimensional bioreactors, we applied cyclic axial load
to the tissue-engineered constructs. Our study showed
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that the material properties of human allograft tissue-engineered constructs were significantly enhanced byreseeding and dynamic conditioning with a bioreactor.

Finally, the ultimate goal would be to use this tissue-engineered human flexor tendon material in a pilotcohort of patients with massive extremity injuries re-quiring multiple tendon grafts. We have received Fed-eral funding for this study and have obtained institu-tional review board approval. Currently, we areworking on collaborations with good manufacturingprocesses facilities to produce this human tendon prod-uct for a clinical trial comparing reconstruction of theflexor digitorum profundus tendons with tendon au-tografts versus reconstruction of the redundant flexordigitorum superficialis tendons with tissue-engineeredflexor tendons.

COMMON PRINCIPLES IN FLEXOR TENDONREPAIR AND REGENERATION RESEARCHIn the past 12 years, we have focused on 2 fundamentalflexor tendon problems facing hand surgeons: postre-pair adhesions and the need for more tendon graftmaterial. We have used molecular biology and tissue-engineering techniques to understand the biomolecularmechanisms regulating tenocyte biology. In the pro-cess, we have gained an understanding of the cells,growth factors, and tissue properties controlling flexortendon wound healing and regeneration. Techniqueshave included the use of natural inhibitors, stem cells,XY fluorescent in situ hybridization, and cell-tissuebioreactors.

Several recurring principles have become evident inthis research. Flexor tendons have great inherentstrength owing to the tightly knit pattern of longitudinalcollagen fibers. The internal endotenon tenocytes havethe capacity to contribute to repair, but most healingoccurs via the epitenon tenocytes and the surroundingsheath. Like wound healing throughout the body, evo-lution has favored fast deposition of collagen over fineremodeling. Modulation of repair should therefore bedirected toward limiting extrinsic healing by inhibitinggrowth factors and other signaling pathways. As wedesign neotendons, we may take advantage of the in-herent strength of native tendons while adding cells thatwill enhance regeneration. All reconstruction, includingtissue-engineered tendon grafting, must have maximalinitial strength to allow early postoperative mobiliza-tion.

As we transition these molecules and products fromthe laboratory to the operating room, additional princi-ples apply. Whether it is an inhibitor molecule or a
tissue-engineered tendon, the product must be simple
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and safe. Although gene therapy strategies via plasmidsor retroviruses have had interesting effects in validanimal models, it is unlikely that the Food and DrugAdministration and other regulatory agencies wouldallow its widespread use in hand surgery. Marketablegrowth factor inhibitors would most likely come fromthe naturally occurring inert molecules described in thisreview. Similarly, for tissue-engineered tendons theproduct will be more marketable if it is easily stored andtransportable and biologically compatible from patientto patient. We look forward to addressing these chal-lenges in the next phase of work in the years to come.

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studies in flexor tendon reconstruction: biomolecular modulation of tendon repair and tissue...

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