Tendon allograft sterilized by peracetic acid/ethanol combined with gamma irradiation

Background

Research and clinical applications have demonstrated that the effects of tendon allografts are comparable to those of autografts when reconstructing injured tendons or ligaments, but allograft safety remains problematic. Sterilisation could eliminate or decrease the possibility of disease transmission, but current methods seldom achieve satisfactory sterilisation without affecting the mechanical properties of the tendon.

Hypothesis

Peracetic acid-ethanol in combination with low-dose gamma irradiation (PE-R) would inactivate potential deleterious microorganisms without affecting mechanical and biocompatible properties of tendon allograft.

Study design

Controlled laboratory design.

Methods

HIV, PPV, PRV and BVDV inactivation was evaluated. After verifying viral inactivation, the treated tendon allografts were characterised by optical microscopy, scanning electron microscopy and tensile testing, and the cytocompatibility was assessed with an MTT assay and by subcutaneous implantation.

Results

Effective and efficient inactivation of HIV, PPV, PRV and BVDV was observed. Histological structure and ultrastructure were unchanged in the treated tendon allograft, which also exhibited comparable biomechanical properties and good biocompatibility.

Conclusion

The preliminary results confirmed our hypothesis and demonstrated that the PE-R tendon allograft has significant potential as an alternative to ligament/tendon reconstruction.

Clinical relevance

Tendon allografts have been extensively used in ligament reconstruction and tendon repair. However, current sterilisation methods have various shortcomings, so PE-R has been proposed. This study suggests that PE-R tendon allograft has great potential as an alternative for ligament/tendon reconstruction.

What is known about this subject

Sterilisation has been a great concern for tendon allografts. However, most sterilisation methods cannot inactivate viruses and bacteria without impairing the mechanical properties of the tendon allograft.

What this study adds to existing knowledge

Peracetic acid/ethanol with gamma irradiation can effectively inactivate viruses and bacteria. Meanwhile, tendon allografts sterilised by this method maintain their physiological tendon structure, biomechanical integrity and good compatibility.     

Comparison of bovine collagen xenografts to autografts in the rabbit

The use of bovine tendon as a xenograft material in humans is attractive because of its ready availability and favorable mechanical characteristics. Previous research has shown that the fibroblasts and some extracellular proteoglycans and glycoproteins, not the collagen matrix itself, in bovine tendon are primarily responsible for its antigenicity. Various attempts have been made to decrease the antigenicity of these grafts. A chloroform/methanol (CM) extraction procedure has been developed that selectively removes the fibroblasts from bovine tendon without destroying the collagen matrix. The mechanical, immunologic, and local host tissue responses to these grafts were compared to autografts and to untreated and glutaraldehyde-treated bovine tendon xenografts. The humoral immune response to a purified bovine Type I collagen product was also studied. The central two-thirds of a rabbit Achilles tendon were replaced with a reversed autograft or an experimental graft. Histologic examination of one- and two- week specimens showed an acute inflammatory response to all grafts. Untreated grafts stimulated a severe inflammatory response and were almost completely resorbed by two weeks. Glutaraldehyde-treated grafts were encapsulated. Cellular repopulation was minimal and inflammatory response was more persistent than in the autograft and CM groups. Inflammatory response to CM-treated grafts was similar to that of autografts. The CM grafts repopulated rapidly with host cells. The mechanical strength of CM grafts was equal to autograft controls at 12 weeks. The mechanical strength of untreated and glutaraldehyde-treated grafts was significantly lower. Measurement of the humoral immune response to these grafts was conducted in an independent group of animals using an enzyme-linked immunosorbent assay. A significant antibody response to untreated, glutaraldehyde-fixed, and CM-treated grafts was detected at 30 days. Antibody titers to glutaraldehyde-fixed and untreated grafts remained elevated at 60 and 90 days. In the CM group, antibody titers decreased to the level of autograft controls by 90 days. No significant antibody response was detected toward purified bovine Type I collagen.     

Mechanical and functional properties of implanted freeze-dried flexor tendons

Tendons harvested from donor dogs were freeze-dried and implanted as free allografts in the paws of recipient experimental animals; the tendons were subsequently tested for mechanical properties, clinical function, and histologic appearance. Free tendon autografts, normal tendons in the operated experimental paw, and normal tendons in the unoperated paw were used as controls and compared with the allografts. Mechanically, the free tendon allografts and autografts were similar. The ultimate strength of the two grafts was statistically the same but significantly less, about one-third, than that of normal tendons. The implanted allografts appeared to be tolerated well by the host and to allow flexor tendon function similar to that allowed by autografts. The histologic appearance of the allograft and autograft tendons was similar at three and six months. Freeze-dried flexor tendon allografting is a satisfactory alternative to free tendon autografting.     

Improved Tendon Radioprotection by Combined Cross-linking and Free Radical Scavenging

Allograft safety is a great concern owing to the risk of disease transmission from nonsterile tissues. Radiation sterilization is not used routinely because of deleterious effects on the mechanical integrity and stability of allograft collagen. We previously reported several individual cross-linking or free radical scavenging treatments provided some radioprotective effects for tendons. We therefore asked whether a combination of treatments would provide an improved protective effect after radiation exposure regarding mechanical properties and enzyme resistance. To address this question we treated 90 rabbit Achilles tendons with a combination of cross-linking (1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide [EDC]) and one of three scavenging regimens (mannitol, ascorbate, or riboflavin). Tendons then were exposed to one of three radiation conditions (gamma or electron beam irradiation at 50 kGy or unsterilized). Combination-treated tendons (10 per group) had increases in mechanical properties and higher resistance to collagenase digestion compared with EDC-only and untreated tendons. Irradiated tendons treated with EDC-mannitol, -ascorbate, and -riboflavin combinations had comparable strength to native tendon and had averages of 26%, 39%, and 37% greater, respectively, than those treated with EDC-only. Optimization of a cross-linking protocol and free radical scavenging cocktail is ongoing with the goal of ensuring sterile allografts through irradiation while maintaining their structure and mechanical properties.     

An in vitro comparison of human flexor and extensor tendon cells

This study examined the in vitro phenotype of cells cultured from both flexor and extensor tendons. Matrix proteins secreted by tendon cells were examined, together with their response to mechanical strain, using cells from the two types of tendon. Immunocytochemical staining using specific antibodies to matrix proteins demonstrated that flexor tendon cells synthesised only three of the five proteins stained for, whilst extensor tendon cells synthesised all five. Gel electrophoresis (used to separate mixtures of proteins) demonstrated that protein secretion was similar for both cell types. Both cell types showed similar patterns of synthesis for total collagen and total protein over time. No difference was found in the response to cyclical strain of cells from the two types of tendon; both cell proliferation and collagen synthesis were stimulated. The study demonstrated that cells obtained from two different types of tendon behave similarly when exposed to identical environmental conditions in vitro.     

The effect of ultrasound on the healing of repaired cockerel tendon: Is collagen crosslinkage a factor?

The acid solubility of the collagen of cockerel tendon demonstrates a similar degree of intermolecular acid-labile cross-linkage to that found in adult human tendon, implying similar maturity of the collagen. By contrast, rabbit tendon collagen demonstrates a dissimilar degree of cross-linkage suggesting immaturity of the collagen. Since this cross-linkage is likely to be related to mechanical properties, the cockerel may well be a better animal model than the rabbit for the study of tendon healing. The application of ultrasound to sutured cockerel tendons produced no change in the mechanical strength of the tendon at six weeks, or its propensity to form adhesions, relative to untreated control sutured tendons. This contrasts with the decrease in strength after ultrasound found by previous workers using rabbits.     

Effect of gamma irradiation on remodeling process of tendon allograft

Freeze-dried tendon allograft sterilized with gamma irradiation could be a reasonable option for ligament substitute. In the current study, the effects of freezing or freeze-drying followed by gamma irradiation on remodeling were analyzed biomechanically in a rat patellar tendon transplantation model at the time of harvest and during a 24-week healing period. The grafts were divided into four groups: fresh-freezing, freeze-drying, fresh-freezing followed by gamma irradiation, and freeze-drying followed by gamma irradiation. Before transplantation, the fresh-frozen grafts and the freeze-dried grafts showed significantly greater tensile strength than the gamma-irradiated grafts. However, at 4 weeks, the tensile strength of each group decreased to the equivalent level, which was approximately 20% of the tensile strength at Time 0, then increased gradually with time. At 24 weeks, the mean tensile strength of each transplanted graft achieved as much as 50% of the tensile strength at Time 0. The change in the tangent modulus with time followed a similar pattern as changes in the tensile strength. This study assumed that the extraarticular tendon transfer model was suitable for evaluating anterior cruciate ligament graft healing. These data suggest that the freeze-dried tendon allografts sterilized by gamma radiation could be a suitable substitute for anterior cruciate ligament reconstruction, if care is taken to protect the graft during the early stage after transplantation (< 4 weeks).     

In vitro study of the role of various preservation methods on mechanical properties of allografts of the patellar tendon]

Tendon allografts are presently used for surgical reconstruction of the anterior cruciate ligament. The aim of this study was the in vitro evaluation of the modification of the mechanical properties of conserved human patellar tendons, deep-frozen or freeze-dried and sterilized with gamma rays. Thirty pairs of patellar tendons (central third) with their bony attachments were removed from fresh corpses, frozen in liquid nitrogen, then conserved at -80 degrees C. Out of each pair, one tendon was submitted to an additional treatment and the other was used as a control. Three types of treatment were studied: 2.5 Mrad irradiation (groupe I), freeze-drying with 1% residual humidity (groupe II), consecutive freeze-drying and irradiation (group III). After warming up (controls and group I treated tendons), the specimens were submitted to mechanical tests on the pulling machine with a suitable arrangement. Two types of tests were performed: a creep test (80 cycles at 1 Hz between 0 and 50 N) then an ultimate tensile strength test (0.25 mm/s). Freeze-drying causes macroscopic morphological alterations, which are even more marked if irradiation is applied in addition. The three types of treatment induce an increase in creep and a decrease in the ultimate, tensile strength and in Young's module, all being statistically significant. The three types of treatment alter the mechanical properties of the allograft. Freeze-drying maintains a better resistance of the graft. The combination of freeze-drying and irradiation is the most damaging method, and freeze-drying even seems to potentialize the harmful effects of irradiation resistance (group II freeze-dried grafts), the treated allografts present with changes in their viscoelastic properties that are incompatible with their clinical use. This study has also shown that some parameters influence the extent of the damaging effects of these three treatments, and experiments are being continued to optimize the conservation and sterilization procedure.     

Cyclic mechanical stimulation rescues achilles tendon from degeneration in a bioreactor system

Physiotherapy is one of the effective treatments for tendinopathy, whereby symptoms are relieved by changing the biomechanical environment of the pathological tendon. However, the underlying mechanism remains unclear. In this study, we first established a model of progressive tendinopathy‐like degeneration in the rabbit Achilles. Following ex vivo loading deprivation culture in a bioreactor system for 6 and 12 days, tendons exhibited progressive degenerative changes, abnormal collagen type III production, increased cell apoptosis, and weakened mechanical properties. When intervention was applied at day 7 for another 6 days by using cyclic tensile mechanical stimulation (6% strain, 0.25 Hz, 8 h/day) in a bioreactor, the pathological changes and mechanical properties were almost restored to levels seen in healthy tendon. Our results indicated that a proper biomechanical environment was able to rescue early‐stage pathological changes by increased collagen type I production, decreased collagen degradation and cell apoptosis. The ex vivo model developed in this study allows systematic study on the effect of mechanical stimulation on tendon biology. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1888–1896, 2015.     

Collagen modifications

Collagen, the major structural protein found in all animal tissues, has a variety of uses in the armamentarium of the plastic surgeon. This article discusses the biologic characteristics of collagen that determine its behavior as an implant material. Clinical applications discussed include hemostatic collagen fleece, collagen sponge wound dressing, composite tissue tendon allografts, injectable collagen, and manipulation of scar collagen.     

Tendon Biomechanics and Crimp Properties Following Fatigue Loading Are Influenced by Tendon Type and Age in Mice

In tendon, type-I collagen assembles together into fibrils, fibers, and fascicles that exhibit a wavy or crimped pattern that uncrimps with applied tensile loading. This structural property has been observed across multiple tendons throughout aging and may play an important role in tendon viscoelasticity, response to fatigue loading, healing, and development. Previous work has shown that crimp is permanently altered with the application of fatigue loading. This opens the possibility of evaluating tendon crimp as a clinical surrogate of tissue damage. The purpose of this study was to determine how fatigue loading in tendon affects crimp and mechanical properties throughout aging and between tendon types. Mouse patellar tendons (PT) and flexor digitorum longus (FDL) tendons were fatigue loaded while an integrated plane polariscope simultaneously assessed crimp properties at P150 and P570 days of age to model mature and aged tendon phenotypes (N = 10–11/group). Tendon type, fatigue loading, and aging were found to differentially affect tendon mechanical and crimp properties. FDL tendons had higher modulus and hysteresis, whereas the PT showed more laxity and toe region strain throughout aging. Crimp frequency was consistently higher in FDL compared with PT throughout fatigue loading, whereas the crimp amplitude was cycle dependent. This differential response based on tendon type and age further suggests that the FDL and the PT respond differently to fatigue loading and that this response is age-dependent. Together, our findings suggest that the mechanical and structural effects of fatigue loading are specific to tendon type and age in mice. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:36–42, 2020     

Mechanical,compositional, and structural properties of the mouse patellar tendon with changes in biglycan gene expression

Tendons have complex mechanical properties that depend on their structure and composition. Some studies have assessed the role of small leucine‐rich proteoglycans (SLRPs) in the mechanical response of tendon, but the relationships between sophisticated mechanics, assembly of collagen and SLRPs have not been well characterized. In this study, biglycan gene expression was varied in a dose dependent manner using biglycan null, biglycan heterozygote and wild type mice. Measures of mechanical (tension and compression), compositional and structural changes of the mouse patellar tendon were evaluated. Viscoelastic, tensile dynamic modulus was found to be increased in the biglycan heterozygous and biglycan null tendons compared to wild type. Gene expression analyses revealed biglycan gene expression was closely associated in a dose‐dependent allelic manner. No differences were seen between genotypes in elastic or compressive properties or quantitative measures of collagen structure. These results suggest that biglycan, a member of the SLRP family, plays a role in tendon viscoelasticity that cannot be completely explained by its role in collagen fibrillogenesis. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1430–1437, 2013     

Mechanical Properties of Reconstructed Achilles Tendon with Transfer of Peroneus Brevis or Flexor Hallucis Longus Tendon

Treatment of chronic Achilles tendon ruptures can be technically difficult because of tendon retraction, atrophy, and short distal stumps. Surgical repair of chronic Achilles tendon ruptures focuses on local and free tendon transfers, as well as reconstruction with allografts or synthetic materials. This study examined the in vitro mechanical properties of a reconstructed Achilles tendon with the peroneus brevis or the flexor hallucis longus tendons in a human cadaver model. The tendons were harvested from 17 fresh-frozen human cadavers, and the same techniques were used for all of the model reconstructions. Biomechanical measurements included the failure load, stiffness, energy-to-peak load, and mode of failure. The mean failure load was significantly higher in the peroneus brevis group (P = .036), and there was no significant difference in stiffness and energy-to-peak load between the peroneus brevis and flexor hallucis longus groups. In every case, the mode of failure involved the tendon graft pulling through either the distal or proximal stump of the Achilles tendon. The greater failure loads observed with the use of peroneus brevis may not be clinically relevant, however, because of the magnitude of the peak loads observed in the cadaveric model. The present study supports the use of either peroneus brevis or flexor hallucis longus for reconstruction of chronic Achilles tendon ruptures and indicates the need for surgeons to carefully reinforce the attachment of the transferred tendon grafts to the stumps of the Achilles tendon to prevent pullout.     

Radioprotection of Tendon Tissue via Crosslinking and Free Radical Scavenging

Ionizing radiation could supplement tissue bank screening to further reduce the probability of diseases transmitted by allografts if denaturation effects can be minimized. It is important, however, such sterilization procedures be nondetrimental to tissues. We compared crosslinking and free radical scavenging potential methods to accomplish this task in tendon tissue. In addition, two forms of ionizing irradiation, gamma and electron beam (e-beam), were also compared. Crosslinkers included 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glucose, which were used to add exogenous crosslinks to collagen. Free radical scavengers included mannitol, ascorbate, and riboflavin. Radioprotective effects were assessed through tensile testing and collagenase resistance testing after irradiation at 25 kGy and 50 kGy. Gamma and e-beam irradiation produced similar degenerative effects. Crosslinkers had the highest strength at 50 kGy, EDC treated tendons had 54% and 49% higher strength than untreated, for gamma and e-beam irradiation respectively. Free radical scavengers showed protective effects up to 25 kGy, especially for ascorbate and riboflavin. Crosslinked samples had higher resistance to collagenase and over a wider dose range than scavenger-treated. Of the options studied, the data suggest EDC precrosslinking or glucose treatment provides the best maintenance of native tendon properties after exposure to ionizing irradiation. One or more of the authors (MGD) have received funding from the Musculoskeletal Transplant Foundation Peer Reviewed Scientific Grants Program (January–December 2005).     

Dose-dependent response of gamma irradiation on mechanical properties and related biochemical composition of goat bone-patellar tendon-bone allografts

We studied the effects of gamma irradiation on the dimensions, mechanical and material properties, and mature hydroxypyridinium crosslink density of collagen in goat patellar tendon-bone specimens. Left and right patellar tendon-bone units were removed from 10 adult female goats and were bisected longitudinally. Each tendon half was frozen, and then the left halves were exposed to 4, 6, or 8 Mrad (40,000,60,000, or 80,000 Gy) of gamma irradiation. The contralateral tendon halves served as controls (no irradiation). Each specimen then was loaded to failure in tension, and its soft-tissue midsubstance was processed to measure collagen content and hydroxypyridinium crosslink density. Dose-dependent reductions in the mechanical properties were found, including 46% (p < 0.01) and 18% (p < 0.05) reductions in maximum force and stiffness, respectively, at 4 Mrad. Similar reductions were noted in material properties, including 37% (p < 0.005) and 8% (p > 0.05) reductions in maximum stress and modulus, respectively, at 4 Mrad. These results are consistent with our previous report involving 2 and 3 Mrad (20,000 and 30,000 Gy) of exposure. We also found significant decreases in hydroxypyridinium crosslink density with 6 Mrad of irradiation (p < 0.05). However, since only one biomechanical parameter (modulus) correlated significantly with only one biochemical measure (hydroxypyridinium crosslink density) (p < 0.05), other possible mechanisms also are being explored to more fully explain these dose-dependent changes.     

自体和异体滑膜内、外肌腱移植的实验研究

目的　比较自体和异体滑膜内肌腱 (intrasynovial tendon,IT )及滑膜外肌腱 (extrasynovialtendon,ET)作鞘管内移植后两者愈合过程和粘连的异同。方法　应用兔新鲜自体和深低温冻存的异体趾深屈肌腱 (IT)、腓骨长肌腱 (ET) ,分别移植于兔左后肢第二趾 (IT)、四趾 (ET)鞘管内修复趾深屈肌腱缺损。自体、异体组各 2 1只兔。术后 10天、3、6周取两组移植腱及对侧正常腱作组织学观察 ;术后 4、8周取材作生物力学测定。结果　两组 IT移植后粘连轻 ,而两组 ET移植后粘连明显 ,滑动功能差于 IT移植(F =14.10 ,P <0 .0 1)。术后 8周时异体移植腱的最大抗断裂载荷值低于自体移植腱 (F =10 .11,P <0 .0 1)。结论　鞘管内自体或异体肌腱移植后均有供腱的组织特异性 ,两者 IT移植后滑动功能均优于 ET组。异体肌腱 (IT、ET)移植后的愈合过程慢于自体移植组。     

The novel use of resorbable Vicryl mesh for in vivo tendon reconstruction to a metal prosthesis

We examined the mechanical properties of Vicryl (polyglactin 910) mesh in vitro and assessed its use in vivo as a novel biomaterial to attach tendon to a hydroxyapatite-coated metal implant, the interface of which was augmented with autogenous bone and marrow graft. This was compared with tendon re-attachment using a compressive clamp device in an identical animal model. Two- and four-ply sleeves of Vicryl mesh tested to failure under tension reached 5.13% and 28.35% of the normal ovine patellar tendon, respectively. Four-ply sleeves supported gait in an ovine model with 67.05% weight-bearing through the operated limb at 12 weeks, without evidence of mechanical failure. Mesh fibres were visible at six weeks but had been completely resorbed by 12 weeks, with no evidence of chronic inflammation. The tendon-implant neoenthesis was predominantly an indirect type, with tendon attached to the bone-hydroxyapatite surface by perforating collagen fibres.     

Regeneration of Achilles tendon with a collagen tendon prosthesis. Results of a one-year implantation study

We previously reported on the short-term biocompatibility of a reconstituted type-I collagen prosthesis that had been tested in the Achilles tendons of rabbits. Preliminary results indicated that, by ten weeks after implantation, carbodiimide-cross-linked implants had been replaced by neotendon in a manner that was similar to that of autogenous tendon grafts that had been used as controls. Also by ten weeks after implantation, glutaraldehyde-cross-linked collagen implants were encapsulated and appeared to have caused an acute inflammatory response. In the present study, carbodiimide and glutaraldehyde-cross-linked collagen implants and autogenous grafts that served as controls were implanted for fifty-two weeks as a replacement for a three-centimeter section of the Achilles tendon of rabbits. The absence of a crimp in a cross-linked implant and the presence of a crimp in normal tendon and in tendon that formed after an implant had been resorbed made it possible to distinguish between a cross-linked implant and new host tendon that had replaced the implant after it was resorbed. New collagen that had replaced the implant and autogenous (control) tendon graft were compared with normal Achilles tendon with respect to the angle and length of the crimp. The autogenous grafts and the carbodiimide-cross-linked collagen implants had been completely resorbed and replaced by neotendon. The neotendon that was present fifty-two weeks after implantation was similar, but not identical, to normal tendon. In contrast, the glutaraldehyde-cross-linked implant was essentially inert, had not been resorbed, and was surrounded by a capsule of collagenous connective tissue. The neotendon in the capsule was also similar, but not identical, to normal tendon. There were more cells in the capsule than in the autogenous grafts or in the carbodiimide-cross-linked implants. The results of the present study indicate that rapid repair is achieved with a carbodiimide-cross-linked collagenous implant that has a structure and mechanical properties that are similar to those of an autogenous tendon graft and that biodegrades at a similar rate. Prolonged biodegradation of a glutaraldehyde-cross-linked collagenous implant results in formation of a capsule and only limited formation of neotendon.     

Carbon Fibres and Plasma‐Preserved Tendon Allografts for Gap Repair of Flexor Tendon in Bovines: Gross,Microscopic and Scanning Electron Microscopic Observations

The efficacy of carbon fibres and plasma‐preserved tendon allografts for gap repair in the superficial digital flexor tendon in the mid‐metatarsal region was evaluated in 12 crossbred calves. Experimental tenectomies were performed, followed by implantation of carbon fibres in group I (12 legs) and plasma‐preserved tendon allografts in group II (12 legs). Gross observations in group I showed filling of the defect with granulation tissue with more vascularity on day 7, which was less prominent at day 14. On day 30, the neotendon formed was slightly thicker and comparable to normal tendon in appearance and texture. On day 90, it exhibited all the characteristics of a fully developed tendon. Whereas, in group II increased vascularity at the site and encapsulation of the graft with connective tissue in early periods was observed. The gap between graft and host was filled with fibrous connective tissue. Peritendinous adhesions were maximum on day 7 which were gradually reduced in both groups. Microscopically, an acute inflammatory reaction in the periphery of carbon fibres was observed on day 7. Immature fibroblasts were arranged in a haphazard pattern at this stage. By day 14, numerous newly formed capillaries and comparatively more mature fibroblasts were present in between and around the carbon fibres which were aligning parallel to the longitudinal axis of the tendon. By day 30 the healing tissue exhibited longitudinal orientation of collagen fibres and was at a more advance stage of maturation. By day 90, the neotendon formed simulated the picture of normal tendon. In the grafted tendon group, there was normal healing tissue at the functional sites between host and grafted tendon. The fibroblastic activity appeared to be both extrinsic and intrinsic in origin. The connective tissue had invaded the graft to a variable distance and there was resorption of graft which was replaced by newly formed connective tissue on day 90. Scanning electron microscopic observation revealed formation of neotendon between carbon fibre strands, resulting in thickening of the implant. In later stages parallel collagen fibres resembling normal tendon were observed in both groups.