Preliminary development of a collagen-PLA composite for ACL reconstruction

Preliminary Development of a Collagen–PLA Compositefor ACL Reconstruction

MICHAEL G. DUNN, LISA D. BELLINCAMPI, ALFRED J. TRIA, JR., JOSEPH P. ZAWADSKY

Orthopaedic Research Laboratory, Division of Orthopaedic Surgery, University of Medicine and Dentistryof New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903

Received 3 August 1995; accepted 9 November 1995

ABSTRACT: Our laboratory is developing resorbable composite implants for reconstruc-tion of the anterior cruciate ligament (ACL) of the knee. Composites were fabricatedby embedding parallel collagen fibers within a poly(lactic acid) (PLA) or collagenmatrix. The mechanical properties, resorption rates, and subcutaneous tissue reactionswere determined for both types of composites. The tensile strength and modulus ofcollagen–PLA composites were twice that of collagen–collagen composites. Subcutane-ous fibrous tissue ingrowth was improved and implant resorption was slightly delayedin the collagen–PLA composites. ACL reconstruction surgeries were performed in rab-bits using collagen–PLA composite implants. After 4 weeks, neoligament tissue wasobserved in seven of eight implants; however, four neoligaments had ruptured eitherin the midsubstance (n Å 2) or at the bone tunnel interface (n Å 2). These results andour previous work suggest that resorbable polymeric composite scaffolds are potentiallyuseful for ACL reconstruction if the implants can be protected from excessive mechani-cal loading during formation of host neoligament tissue. q 1997 John Wiley & Sons, Inc.J Appl Polym Sci 63: 1423–1428, 1997

Key words: anterior cruciate ligament; collagen fibers; poly(lactic acid); tissue engi-neering; biomaterial; composite; scaffolds

INTRODUCTION oping resorbable biomaterials for use as scaffoldson which cells synthesize neoligament tissue toreplace the ACL. The ideal scaffold would provideInjury to the anterior cruciate ligament (ACL) ofhigh strength initially, then gradually degrade,the knee can result in disability and progressivetransferring mechanical loads to neoligament tis-degeneration of other structures in the joint. Asue (Fig. 1). This ‘‘tissue engineering’’ approachruptured ACL must be surgically reconstructed tois potentially useful for regeneration of a varietyrestore normal joint function, as primary repairof tissues and organs.3has a high failure rate. Although ACL anatomy,

Scaffolds for tissue engineering applicationsstructure, biomechanics, and healing have beencan be natural extracellular matrix-derived bio-extensively studied, there is still no biologicalpolymers (such as collagen), synthetic resorbablegraft or permanent prosthesis ideally suited forpolymers (such as aliphatic polyesters), or hybridACL reconstruction.1,2 Our laboratory is devel-combinations of natural and synthetic polymers.Composite collagenous scaffolds (collagen fibers

Correspondence to: M. G. Dunn at UMDNJ-Robert Wood in a collagen matrix) can induce neotendon forma-Johnson Medical School, Orthopaedic Research Laboratory,tion in the Achilles tendon4,5 and neoligament for-Division of Orthopaedics, MEB 424,1 Robert Wood Johnson

Pl-CN 19, New Brunswick, NJ 08903 mation in the ACL of rabbits.6 In our previousContract grant sponsor: Whitaker Foundation ACL reconstruction study,6 about one-half of theContract grant number: 90-0195

collagen composites ruptured before adequateContract grant sponsor: Foundation of UMDNJq 1997 John Wiley & Sons, Inc. CCC 0021-8995/97/111423-06 neoligament tissue was formed, in part due to sur-

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1424 DUNN ET AL.

hydrothermal-cyanamide treatment10 as pre-viously described.7 Fibers were heated to 1107Cfor 3 days under high vacuum (õ0.1 micron Hg),then exposed to a saturated solution of cyanamide(a carbodiimide; Sigma, St. Louis, MO) for 24 hat room temperature.

Fiber bundles were prepared by aligning 200or 500 collagen fibers in parallel. Composites weremade by using either collagen or PLA as the ma-trix around the aligned collagen fibers. Compos-ites contained approximately 50% fiber and 50%matrix (w/w). The collagen matrix was appliedby dipping the collagen fibers within a 1% (w/v)

Figure 1 Ideal strength vs. time profile for a bone– acidic bovine dermal collagen dispersion and air-ligament–bone complex surgically reconstructed using

drying. PLA (Medisorb 100 L, DuPont, Wilming-a resorbable ‘‘scaffold’’ implant. The gradual strengthton, DE) was applied by dipping the collagen fi-loss of the implant is offset by strength gain in thebers in a 10% (w/v) solution of PLA in chloroformneoligament tissue. As a result, the net strength of theand drying under vacuum overnight at room tem-bone–ligament–bone complex remains high and rela-perature (Fig. 2). The PLA11 was 100% poly-L-tively constant.lactide (residual monomer content approximately1%) with a weight-average molecular weight of100/ M, an inherent viscosity of 0.9 dL/g, and agical factors. Our goal was to improve the initialspecific gravity of 1.55. The glass transition andstrength, strength retention, and rate of neoliga-melting temperatures were approximately 55–ment formation associated with resorbable com-607C and 170–1757C, respectively.posites for ACL reconstruction.

The strength and resorption rate of collagenfibers can be improved by varying their diameter7

Mechanical Properties of Compositesand by crosslinking.8 Although extensively cross-linked collagen fibers are very strong, they may Saline-soaked composites (nÅ 10) containing 500resorb too slowly, resulting in chronic inflamma- collagen fibers with either the collagen or PLAtion after implantation.5 Another way to improve matrix were tested in tension to failure on an In-the properties of these composites is to modify stron Model 4204 materials tester (Instron Corp.,the matrix surrounding the collagen fibers. In this Canton, MA). The sample gauge length was 10study, we fabricated collagen fiber-based compos- mm, and the elongation rate was 100 mm per minites using either collagen or a synthetic aliphaticpolyester [poly(lactic acid)9 (PLA)] as the ma-trix. We compared the initial mechanical proper-ties and subcutaneous resorption rates for colla-gen–PLA and collagen–collagen composites. Wealso utilized a surgical model in rabbits to assessthe feasibility of using collagen–PLA compositesfor ACL reconstruction.

MATERIALS AND METHODS

Fabrication of Composites

Collagen fibers (dry diameter 50–70 mm) weremade by extrusion of an acidic 1% (w/v) type Ibovine dermal collagen dispersion into fiber for-mation buffer (pH 7.5, 377C) as previously de- Figure 2 Scanning electron micrograph of the colla-scribed.4–8 Fibers were rinsed in alcohol and dis- gen–PLA composite. The collagen fibers (CF) weretilled water and dried under tension overnight at loosely surrounded by the PLA matrix (bar Å 1,000

mm).room temperature. Fibers were crosslinked by de-


COLLAGEN–PLA COMPOSITE FOR ACL RECONSTRUCTION 1425

(1000% strain/min). Structural properties (peak and through both bone tunnels at the anatomicattachment sites of the removed ACL. The endsload [N], deformation [mm], and stiffness [N/

mm]) were obtained from the load-deformation of the composite were secured (using a 4-0 Prolenesuture) to the periosteum of the femur and thecurves. Material properties (ultimate tensile

strength [MPa], strain [%], and modulus [MPa]) tibia with the composite under tension [see Fig.5(A)]. The patella was reduced, and the joint cap-were determined by normalizing the structural

properties by the dimensions of each sample. sule and skin were closed with 4-0 Prolene sutureusing a running simple stitch. The limb was cov-Cross-sectional areas were measured using Ver-

nier calipers; two perpendicular measurements ered with gauze for several days to protect theincision sites.were made and an elliptical cross section was as-

sumed. Animals were returned to individual cages withunrestricted activity and given food and water adlibitum. Tetracycline was given orally for 2 weeks

Subcutaneous Implants postimplantation to prevent infection. Animalswere sacrificed at 4 weeks postimplantation byComposites for subcutaneous implantation con-

tained 200 collagen fibers and either the collagen general anesthesia followed by intracardiac injec-tion of pentobarbitol sodium (Webster, Sterling,or PLA matrix. Samples of 1 cm length were im-

planted subcutaneously in anesthetized male MA). Neoligament tissue was evaluated by grossobservation and by examination of paraffin-em-New Zealand white rabbits. For all surgeries, NIH

guidelines for the care and use of laboratory ani- bedded, hematoxylin- and eosin-stained histologi-cal slides from neoligament midsubstance andmals were observed.12 Implants were retrieved at

2 and 4 weeks postimplantation (n Å 5). The surgical bone tunnel samples.number of collagen fibers remaining intact wasobtained by analyzing hematoxylin and eosin-

Statistical Analysesstained cross sections of the implants under a Ni-kon light microscope as previously described.13 Analysis of variance was performed using Stat-

graphicst software (Rockville, MD) to determinethe effects of matrix composition (collagen vs.ACL Reconstruction SurgeryPLA) on the initial mechanical properties and

In a previous study, we reconstructed the ACL in subcutaneous resorption rates of the composites.rabbits using collagen fiber–collagen matrix im- Differences between individual groups were con-plants.6 In the present study, eight ACL recon- sidered significant for P õ .05.struction surgeries were performed using collagenfiber–PLA matrix composites in skeletally ma-ture New Zealand white rabbits. Each implant RESULTScontained 500 collagen fibers embedded in thePLA matrix, with PLA plugs on the ends. An 8 in. Mechanical Properties of Compositeslength of 4-0 Prolene suture (Ethicon, Somerville,

Compared to collagen–collagen composites (Fig.NJ) with a straight needle on the end was3), collagen–PLA composites had significantlyattached to each PLA plug for surgical fixationgreater structural properties (breaking load 40of the composite. Prior to surgery, implants were{ 5 Newtons; stiffness 14 { 3 Newtons/mm), andsterilized in ExsporTM chemosterilant (Alcidematerial properties (ultimate tensile strength 13Corp., Norwalk, CT) for 1 h and rinsed in sterile{ 1 MPa; modulus 37 { 9 MPa). The strain atsaline overnight. The animals were anesthetizedfailure was decreased for the collagen–PLA com-and the hind limbs were shaved and prepared forposites.sterile surgery.

All surgeries were performed by an orthopedicsurgeon in our group (A. J. T.) . The ACL was re- Subcutaneous Resorption and Tissue Reactionmoved by sharp dissection at the tibial and femo-ral attachment sites. The fat pad was left intact. The subcutaneous resorption rate of the implants

was based on the number of collagen fibers re-A 1.5 mm-diameter bone tunnel was createdthrough the lateral femoral condyle and the tibia maining intact at 2 and 4 weeks postimplantation

(Fig. 4). Approximately 75% of the implanted col-(exiting at the anatomic ACL attachment sites)using a minidriver drill. A sterile collagen–PLA lagen fibers remained intact at 2 weeks; less than

50% of the implanted collagen fibers remained in-composite (length 4 cm) was placed in the joint


1426 DUNN ET AL.

of the collagen fibers were degraded by 4 weekspostimplantation, while the PLA matrix re-mained partially intact [Fig. 5(C)]. Within thesurgical bone tunnels, new bone and soft tissuewere found around and within the composite im-plants [Fig. 5(D)].

DISCUSSION

Both natural4–6 and synthetic14,15 resorbablepolymers have been used as scaffolds for tendonor ligament reconstruction. Natural, extracellularmatrix-derived polymers such as collagen haveadvantageous biological properties that syntheticFigure 3 Material properties of resorbable compos-polymers lack. On the other hand, synthetic poly-ites, prior to implantation. The ultimate tensilemers are cost-effective, exhibit less batch-to-batchstrength and modulus of the collagen–PLA composites

were significantly greater than for the collagen–colla- variability, and have physicochemical propertiesgen composites. which are readily modified to suit specific applica-

tions. It is likely that hybrid biomaterials combin-ing natural and synthetic polymers will be needed

tact at 4 weeks. At both time periods, the colla- to satisfy the rigorous physical and biological re-gen–PLA composites had more collagen fibers re- quirements for a resorbable ligament reconstruc-maining intact; however, this difference was not tion device. To begin testing this hypothesis, westatistically significant. developed hybrid composites consisting of natural

Histological analysis of retrieved subcutaneous fibers (collagen) embedded within in a syntheticimplants revealed degrading composites sur- matrix (PLA).rounded by fibrous tissue and similar amounts of The ultimate tensile strength of the collagen–inflammatory cells. Collagen–collagen compos- PLA composites (13 MPa) was about one-third ofites were well encapsulated and had limited fi- the strength of the ACL (38 MPa).16 Collagen–brous tissue ingrowth. Collagen–PLA composites PLA composites had significantly greaterappeared to have more fibrous tissue ingrowth strength and modulus than those of the collagen–reaching the center of the implants. Polarized collagen composites. This may be due to thelight microscopy revealed that the PLA network greater strength and modulus of the PLA matrixwas largely intact at 4 weeks postimplantation.

ACL Reconstruction Surgery

In one of the eight operated knees, neoligamenttissue failed to form in response to the collagen–PLA composite implant. In seven of the eight op-erated knees, neoligament formation was inducedby implantation of the collagen–PLA composite(Fig. 5). The neoligament tissue appeared glisten-ing and white, similar to normal ligament tissue[Fig. 5(B)]. Three of the seven neoligaments werecompletely intact between the tibia and the femurwhen the joint was opened at 4 weeks postimplan-tation. Two neoligaments had broken in their mid-substance; two neoligaments had broken at theirinterface with the surgical bone tunnel.

Histological sections from the neoligament Figure 4 Collagen fiber resorption rate following sub-midsubstance showed that the implants were cutaneous implantation in rabbits. The slight delay inlargely degraded and replaced by host neoliga- fiber resorption due to application of the PLA matrix

was not statistically significant.ment tissue and inflammatory cells. Nearly all


COLLAGEN–PLA COMPOSITE FOR ACL RECONSTRUCTION 1427

Figure 5 ACL reconstruction surgery in rabbits: (A) the collagen–PLA compositewas placed through femoral and tibial bone tunnels at the anatomic attachment sitesof the removed ACL; (B) example of intact neoligament tissue (NL; arrow) connectingthe tibia to the femur at the anatomic attachment sites of the surgically removedACL; (C) neoligament tissue was composed of host fibrous and inflammatory tissueinfiltrating the degrading composite scaffold (* Å PLA; bar Å 25 mm); (D) in the bonetunnel, new bone (NB) and osteoid tissue infiltrated the degrading composite scaffold(* Å PLA; bar Å 50 mm).

(57 and 2100 MPa, respectively)11 compared to strength retention in this study. Since the PLAmatrix was still largely intact after 4 weeks im-the uncrosslinked collagen matrix (5 and 10 MPa,

respectively).7 It is also possible that the PLA ma- plantation, the strength retention profile may beimproved for collagen–PLA composites comparedtrix limits wetting and swelling of the collagen

fibers. We previously found that dry collagen fi- to collagen–collagen composites. Studies are un-derway to determine whether PLA (or other syn-bers have about three to five times the strength

of wet collagen fibers, depending on the fiber di- thetic matrix materials) improve the strength re-tention profile for collagen fiber-based implantsameter and crosslinking method.7

Histological evaluation of subcutaneous im- in vitro and in vivo.In our short-term ACL reconstruction study,plants at 2 or 4 weeks indicated that more colla-

gen fibers remained intact within the PLA matrix the ligament was completely removed and re-placed by a collagen–PLA composite which in-compared to the collagen matrix; however, this

difference in fiber mass resorption was not statis- duced neoligament formation in seven of eighttreated knees (after excision, the rabbit ACL istically significant. We did not measure composite


1428 DUNN ET AL.

and Medical Devices. Presented in part at the 39th An-incapable of spontaneous regeneration).17 Onlynual Meeting of the Orthopaedic Research Societythree of the seven neoligaments, however, were(1993) and the 3rd World Biomaterials Congresscompletely intact when the animals were sacri-(1992).ficed at 4 weeks postimplantation, consistent with

our previous study.6 Gross and histological obser-vations of explants indicated that the mode of fail-

REFERENCESure was most likely due to excessive loading of thejoint or mechanical shearing where the implants

1. M. G. Dunn, in Ligaments of the Knee, A. J. Tria,emerged from the bone tunnels. Thus, it is likelyJr., Ed., Churchill Livingstone, New York, 1995,that the performance of these composites can beChap. 13.enhanced by smoothing sharp bone tunnel edges

2. M. G. Dunn and S. H. Maxian, in Implantation Bi-and protecting the reconstructed knee from exces- ology: The Host Response and Biomedical Devices,sive loads. This can be accomplished by temporar- R. S. Greco, Ed., CRC Press, Boca Raton, FL, 1994,ily or partially immobilizing the treated knee and Chap. 13.leaving the untreated leg to bear mechanical 3. R. Langer and J. P. Vacanti, Science, 260, 920–926loads. (1993).

4. A. J. Wasserman, Y. P. Kato, D. Christiansen,Results of this study and our previousM. G. Dunn, and F. H. Silver, Scan. Microsc., 3,work 6–8,13,18 suggest that ACL reconstruction1183–1200 (1989).using a resorbable device is feasible; however,

5. Y. P. Kato, M. G. Dunn, J. P. Zawadsky, A. J. Tria,the resorbable composites described here areand F. H. Silver, J. Bone Jt. Surg., 73A, 561–574not optimal for ACL reconstruction. For exam-(1991).ple, we no longer use dehydrothermal treat-

6. M. G. Dunn, A. J. Tria, J. R. Bechler, R. S. Ochner,ment to crosslink collagen fibers, since we J. P. Zawadsky, Y. P. Kato, and F. H. Silver, Am.showed that ultraviolet light crosslinked colla- J. Sports Med., 20, 507–515 (1992).gen fibers are equally strong 8 and much more 7. M. G. Dunn, P. N. Avasarala, and J. P. Zawadsky,stable in the presence of nonspecific proteolytic J. Biomed. Mater. Res., 27, 1545–1552 (1993).enzymes.19 Furthermore, PLA is not the ideal 8. K. S. Weadock, E. J. Miller, L. D. Bellincampi, J. P.

Zawadsky, and M. G. Dunn, J. Biomed. Mater.polymer for this application for several rea-Res., 29, 1373–1379 (1995).sons: collagen and PLA were difficult to com-

9. M. Vert, S. M. Li, G. Spenlehauer, and P. Guerin,bine, probably due to the relative hydropho-J. Mater. Sci. Mater. Med., 3, 432–446 (1992).bicity of PLA. Scanning electron microscopy re-

10. K. S. Weadock, R. M. Olson, and F. H. Silver, Bio-sults suggested poor bonding between collagenmat. Med. Dev. Artif. Organs, 11, 293–318 (1984).fibers and the PLA matrix. Other polymeric

11. MedisorbTM Bioresorbable Polymers. Properties,matrices may provide improved fiber–matrix Uses, Storage, and Handling, DuPont Co., Wilming-interfacial bonding and higher composite ton, DE, 1989.strengths. Finally, recent reports suggest that 12. NIH Guide for the Care and Use of Laboratory Ani-in the long term PLA implants may cause re- mals, NIH Publication 85-23, NIH, Bethesda, MD,sorption of bone.20 1985.

13. M. G. Dunn, S. H. Maxian, and J. P. Zawadsky, J.We are currently developing ‘‘second genera-Orthop. Res., 12, 128–137 (1994).tion’’ hybrid devices combining ultraviolet light

14. H. E. Cabaud, J. A. Feagin, and W. G. Rodkey, Am.crosslinked collagen fibers8,19 with other re-J. Sports Med., 10, 259–265 (1982).sorbable polymeric matrices. We also plan to com-

15. S. J. Shieh, M. C. Zimmerman, and J. R. Parsons,bine synthetic resorbable fibers (providing highJ. Biomed. Mater. Res., 24, 789–808 (1990).strength) with extracellular matrix-derived coat-

16. F. R. Noyes, D. L. Butler, E. S. Grood, R. F. Zer-ings (providing biocompatibility for cells)18 to op- nicke, and M. S. Hefzy, J. Bone Jt. Surg., 66A,timize both the bulk and surface properties of re- 344–352 (1984).sorbable composite scaffolds for ACL reconstruc- 17. F. L. Hefti, A. Kress, J. Fasel, and E. W. Morscher,tion. J. Bone Jt. Surg., 73A, 373–383 (1991).

18. M. G. Dunn, J. B. Liesch, M. L. Tiku, and J. P. Za-wadsky, J. Biomed. Mater. Res., 29, 1363–1371Jack Abboudi, MD, assisted with preliminary develop-

ment of collagen–PLA composites. This study was (1995).19. K. S. Weadock, E. J. Miller, E. L. Keuffel, and M. G.funded by grants to M. G. D. from the Whitaker Foun-

dation (#90-0195) and the Foundation of UMDNJ. Dunn, J. Biomed. Mater. Res. 32, 221–226 (1996).20. J. Suganuma and H. Alexander, J. Appl. Bio-L. D. B. was supported by a Summer Research Fellow-

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Preliminary development of a collagen-PLA composite for ACL reconstruction