Fibrocartilage Formation Research Articles

Development of the supraspinatus tendon‐to‐bone insertion: Localized expression of extracellular matrix and growth factor genes

The adult healing response of the rotator cuff tendon-to-bone insertion site differs from the ordered process of insertion site development. Healing is characterized by disorganized scar and a lack of fibrocartilage formation, in contrast to the well organized fibrocartilaginous transition which forms during the normal development of the tendon-to-bone insertion. The purpose of this study was to localize the expression of a number of extracellular matrix and growth factor genes during insertion site development in order to guide future strategies for augmenting adult rotator cuff healing. The rotator cuff was morphologically distinct at 13.5 dpc (days postconception). Neo-tendon was evident as a condensation of cells adjacent to bone. The interface between tendon and bone did not form into a mature fibrocartilaginous insertion until 21-days postnatally, based upon the appearance of four distinct zones with a mineralized humeral head. Fibroblasts of the supraspinatus tendon expressed type I collagen at all timepoints. Type II collagen was first expressed by chondrocytes in the fibrocartilage and mineralized fibrocartilage at 7 days and persisted in the mineralized fibrocartilage at 56 days. Type X collagen was first expressed by the chondrocytes in the mineralized fibrocartilage at 14 days and persisted in the mineralized fibrocartilage at 56 days. A shift from TGF-beta3 to TGF-beta1 expression occurred at 15.5 dpc.

Biologic augmentation of rotator cuff tendon repair

It is known that a histologically normal insertion site does not regenerate after rotator cuff tendon-to-bone repair. Cytokines play an important role in cell chemotaxis, proliferation, matrix synthesis, and cell differentiation and may thus improve rotator tendon-to-bone healing. We have used a sheep infraspinatus repair model to evaluate the effect of osteoinductive growth factors (bone morphogenetic protein [BMP] 2, BMP-7, transforming growth factor [TGF] beta1, TGF-beta2, TGF-beta3, and fibroblast growth factor) and BMP-12 on tendon-to-bone healing. We have found that these molecules improve formation of new bone and fibrocartilage at the healing tendon attachment site, resulting in improved load to failure. Several other avenues have the potential to augment repair site biology. For example, because platelets are known to contain various cytokines, they may be isolated from autologous blood and could provide an effective method by which to deliver growth factors to a rotator cuff tendon repair site. Furthermore, modalities that improve local vascularity, such as low-intensity pulsed ultrasound or nitroglycerine, have the potential to augment rotator cuff repair healing. Finally, important information about the biology of tendon healing can be gained from studies of substances that inhibit healing, such as nicotine and nonsteroidal anti-inflammatory medications.

Decreased muscle loading delays maturation of the tendon enthesis during postnatal development

Physical environment influences the development and maintenance of musculoskeletal tissues. The current study uses an animal model to explore the role of the physical environment on the postnatal development of the supraspinatus tendon enthesis. A supraspinatus intramuscular injection of botulinum toxin A was used to paralyze the left shoulders of mice at birth. The supraspinatus muscles of right shoulders were injected with saline to serve as contralateral controls. The supraspinatus enthesis was examined after 14, 21, 28, and 56 days of postnatal development. Histologic assays were used to examine fibrocartilage morphology and percentage osteoclast surface. Micro-computed tomography was used to examine muscle geometry and bone architecture. At 14 days there were no differences between groups in fibrocartilage formation, muscle geometry, bone architecture, or osteoclast surface. When comparing groups at 21, 28, and 56 days, muscle volume was decreased, fibrocartilage development was delayed, mineralized bone was decreased, and osteoclast surface was higher at each timepoint in the botulinum group compared to the contralateral saline control group. Our results indicate that the development of the tendon enthesis is sensitive to its mechanical environment. A reduction in muscle loading delayed the development of the tendon-to-bone insertion site by impeding the accumulation of mineralized bone. Physical factors did not play a significant role in enthesis maturation in the first 14 days postnatally, implying that biologic factors may drive early postnatal development.

The Role of Bone Morphogenetic Proteins in Rotator Cuff Tendon Repair

Summary: Bone morphogenetic proteins are multi-functional molecules that play an important role in tissue differentiation. The classic BMPs (BMP-2, 4, 7) are osteoinductive, whereas more recently described BMPs (BMP-12, 13) are expressed during tendon insertion site formation in embryogenesis and have been found to induce formation of fibrocartilage. This review examines several studies that have tested the effects of bone morphogenetic proteins in rotator cuff tendon repair models.

Basic Science and Treatment Options for Articular Cartilage Injuries

Articular cartilage injuries can produce significant musculoskeletal morbidity for both young and active aging patient populations. The complex and highly specialized composition of normal hyaline cartilage makes treatment of focal chondral injuries a formidable challenge for the basic scientist, surgeon, and physical therapist. The current array of surgical treatment options offers palliative, reparative, and restorative treatment strategies. Palliative options include simple arthroscopic debridement. Reparative strategies utilize marrow stimulation techniques to induce formation of fibrocartilage within the chondral defect. Restorative tactics attempt to replace damaged cartilage with hyaline or hyaline-like tissue using osteochondral or chondrocyte transplantation. Furthermore, while treatment success is obviously dependent on good surgical selection and technique, the importance of sound, compliant postoperative rehabilitation cannot be understated. The purpose of this article is to review the basic science of articular cartilage, current treatment options available, and outline the clinical decision making involved when using these procedures by presenting the algorithm used at our institution for treating focal cartilage lesions.

Gene Regulation ex Vivo within a Wrap-Around Tendon

This study tested the hypothesis that physiologic tendon loading modulates the fibrous connective tissue phenotype in undifferentiated skeletal cells. Type I collagen sponges containing human bone marrow stromal cells (MSCs) were implanted into the midsubstance of excised sheep patellar tendons. An ex vivo loading system was designed to cyclically stretch each tendon from 0 to 5% at 1.0 Hz. The MSC-sponge constructs were implanted into 2 tendon sites: the first site subjected to tension only and a second site located at an artificially created wrap-around region in which an additional compressive stress was generated transverse to the longitudinal axis of the tendon. The induced contact pressure at the wraparound site was 0.55 +/- 0.12 MPa, as quantified by pressure-sensitive film. An MSC-sponge construct was maintained free swelling in the same bath as an unloaded control. After 2 h of tendon stretching, the MSC-sponge constructs were harvested and real-time PCR was used to quantify Fos, Sox9, Cbfa1 (Runx2), and scleraxis mRNA expression as markers of skeletal differentiation. Two hours of mechanical loading distinctly altered MSC differentiation in the wrap-around region and the tensile-only region, as evidenced by differences in Fos and Sox9 mRNA expression. Expression of Fos mRNA was 13 and 52 times higher in the tensile-only and wrap-around regions, respectively, compared to the free-swelling controls. Expression of Sox9 mRNA was significantly higher (2.5-3 times) in MSCs from the wraparound region compared to those from the tensile-only region or in free-swelling controls. In contrast, expression levels for Cbfa1 did not differ among constructs. Scleraxis mRNA was not detected in any construct. This study demonstrates that the physiologic mechanical environment in the wrap-around regions of tendons provides stimuli for upregulating early response genes and transcription factors associated with chondrogenic differentiation. These differentiation responses begin within as little as 2 h after the onset of mechanical stimulation and may be the basis for the formation of fibrocartilage that is typically found in the wrap-around region of mature tendons in vivo.

Microfracture and bone morphogenetic protein 7 (BMP-7) synergistically stimulate articular cartilage repair

Microfracture is used to treat articular cartilage injuries, but leads to the formation of fibrocartilage rather than native hyaline articular cartilage. Since bone morphogenetic protein 7 (BMP-7) induces cartilage differentiation, we hypothesized that the addition of the morphogen would improve the repair tissue generated by microfracture. We determined the effects of these two treatments alone and in combination on the quality and quantity of repair tissue formed in a model of full-thickness articular cartilage injury in adolescent rabbits. Full-thickness defects were made in the articular cartilage of the patellar grooves of forty, 15-week-old rabbits. Eight animals were then assigned to (1) no further treatment (control), (2) microfracture, (3) BMP-7, (4) microfracture with BMP-7 in a collagen sponge (combination treatment), and (5) microfracture with a collagen sponge. Animals were sacrificed after 24 weeks at 39 weeks of age. The extent of healing was quantitated by determining the thickness and the surface area of the repair tissue. The quality of the repair tissue was determined by grading specimens using the International Cartilage Repair Society Visual Histological Assessment Scale. Compared to controls, BMP-7 alone increased the amount of repair tissue without affecting the quality of repair tissue. Microfracture improved both the quantity and surface smoothness of repair tissue. Compared to either single treatment, the combination of microfracture and BMP-7 increased both the quality and quantity of repair tissue. Microfracture and BMP-7 act synergistically to stimulate cartilage repair, leading to larger amounts of repair tissue that more closely resembles native hyaline articular cartilage.

Arthroplasty of the shoulder

Although the first shoulder arthroplasty was implanted in 1893 by the French surgeon Jules-Emile Pean,[1][1] the development of the procedure came in the 1950s when Neer[2][2] described the results using a vitallium prosthesis to treat comminuted fractures of the head of the humerus. About 20

Rabbit articular cartilage defects treated by allogenic chondrocyte transplantation

Articular cartilage defects have a poor capacity for repair. Most of the current treatment options result in the formation of fibro-cartilage, which is functionally inferior to normal hyaline articular cartilage. We studied the effectiveness of allogenic chondrocyte transplantation for focal articular cartilage defects in rabbits. Chondrocytes were cultured in vitro from cartilage harvested from the knee joints of a New Zealand White rabbit. A 3 mm defect was created in the articular cartilage of both knees in other rabbits. The cultured allogenic chondrocytes were transplanted into the defect in the right knees and closed with a periosteal flap, while the defects in the left knees served as controls and were closed with a periosteal flap alone, without chondrocytes. Healing of the defects was assessed at 12 weeks by histological studies. Allogenic chondrocyte transplantation significantly increased the amount of newly formed repair tissue (P=0.04) compared with that found in the control knees. The histological quality score of the repair tissue was significantly better (P=0.05), with more hyaline characteristics in the knees treated with allogenic chondrocytes than in the control knees. Articular cartilage defects treated with allogenic chondrocyte transplantation result in better repair tissue formation with hyaline characteristics than those in control knees.

Stem cell-coated titanium implants for the partial joint resurfacing of the knee

The goal of the present study was to evaluate the partial surface replacement of the knee with stem cell-coated titanium implants and to provide a basis for a successful treatment of large osteochondral defects. Mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates of adult sheep. Round titanium implants with a diameter of 2×7.3 mm were seeded with autologous MSC and inserted into an osteochondral defect in the medial femoral condyle. As controls, defects received either an uncoated implant or were left untreated. Nine animals with 18 defects were sacrificed after 6 months. Histological evaluation was performed by intravital polychrome fluorescent labelling, intravital perfusion with Indian ink, microradiographs and differential staining with toluidine blue. The quality of regenerated cartilage was assessed by in situ hybridization of collagen type II and immunohistochemistry of collagen types I and II. In 50% of the cases, defects treated with MSC-coated implants showed a complete regeneration of the subchondral bone layer. In these cases collagen type II and only traces of collagen type I were detected. A high level of collagen type II mRNA expression compared to articular cartilage indicates regenerating hyaline-like cartilage. A total of 50% of MSC-coated and uncoated implants failed to osseointegrate and formation of fibrocartilage was observed. Untreated defects as well as defects treated with uncoated implants demonstrated incomplete healing of subchondral bone and formation of fibrous cartilage. A modified histological score according to Wakitani significantly demonstrated better results for cell-coated implants (8.8±6.4) than for uncoated implants (5.5±3.9) and for untreated defects (2.8±2.5). Our results demonstrate that, in a significant number of cases, a partial joint resurfacing of the knee with stem cell-coated titanium implants occur. A slow bone and cartilage regeneration and an incomplete healing in half of the MSC-coated implants are limitations of the presented method. To improve our approach and optimize the experimental parameters, further investigations are needed prior to clinical application.

Ultrastructural Differences in Cranial Cruciate Ligaments from Dogs of Two Breeds with a Differing Predisposition to Ligament Degeneration and Rupture

Cranial (anterior) cruciate ligament (CCL) samples were obtained from dogs of the Labrador retriever (LR) and greyhound (GH) breeds, of which the former but not the latter is predisposed to CCL rupture. Electron microscopy revealed that the collagen fibril diameters of GHs were larger than those of LRs (P=0.03). Histological examination revealed a "fibrocartilaginous" appearance of CCLs in seven of eight GHs, and, to a lesser extent, in three of eight LRs. The formation of fibrocartilage is clearly not a disadvantage to the healthy racing GH, and cannot be regarded as a pathological degeneration in this breed. It is suggested that fibrocartilage is formed as a beneficial physiological adaptation to the compression of CCLs caused by tensile stress as a result of the tightening of two twisted bands. Fibrocartilage would appear to protect CCLs in the GH, but it may be indicative of a mild degenerative change, which may eventually lead to rupture in the LR.

Nonarthroplasty Treatment of Glenohumeral Cartilage Lesions

Treatment of young, active persons with symptomatic cartilage lesions of the glenohumeral joint represents a significant challenge. Diagnosis of glenohumeral chondral defects is not always straightforward and effective treatment requires familiarity with a number of techniques. Low-demand individuals may accept palliative therapy in the form of arthroscopic debridement as a temporizing solution. However, younger, high-demand individuals require a careful, stepwise approach that includes reparative, restorative, and reconstructive strategies. Reparative strategies use marrow-stimulation techniques to induce formation of fibrocartilage. Restorative tactics attempt to replace damaged cartilage with hyaline or hyaline-like tissue using osteochondral or chondrocyte transplantation. Large lesions that are not candidates for reparative or restorative procedures can be approached using reconstruction methods such as biologic resurfacing. This review examines causes of chondral injury in the glenohumeral joint, discusses diagnostic strategies, and presents a practical framework including palliative, reparative, restorative, and reconstructive options with which one can formulate a treatment plan for these patients.

Synovitis in a murine model of human factor VIII deficiency

Recurrent joint bleeding is the most common musculoskeletal manifestation of haemophilia and leads to a target joint and synovitis. The pathobiology of haemophilic synovitis (HS) is not well understood. Here the histopathological changes that occur following haemarthrosis were examined in an animal model for human HS. After two haemarthrosis, there was soft tissue and joint swelling and histological changes of acute synovitis included infiltration of the sub-synovial layer by mononuclear cells and neutrophils, thickening of the synovial membrane with villus formation, and hyperplasia of blood vessels. Subacute changes were evident after three haemarthrosis; muscle atrophy was present and an intense mononuclear cell infiltrate filled the sub-synovial space. There was destruction of articular surfaces and loss of cartilage. Seventeen months after three haemarthrosis, chronic joint changes included gross deformity and loss of congruence due to dense fibrotic tissue filling the joint space. The mononuclear inflammatory cell infiltrate and thickened synovial membrane persisted. Pits and erosions of articular surfaces and sub-chondral cysts were present. There was fibro-cartilage and new bone formation. This model of human HS should be useful to fully evaluate the biochemical and molecular changes that occur following joint bleeding and to test novel therapeutics to prevent HS.

Tissue engineered cartilage on collagen and PHBV matrices

Cartilage engineering is a very novel approach to tissue repair through use of implants. Matrices of collagen containing calcium phosphate (CaP–Gelfix ®), and matrices of poly(3-hydroxybutyric acid- co-3-hydroxyvaleric acid) (PHBV) were produced to create a cartilage via tissue engineering. The matrices were characterized by scanning electron microscopy (SEM) and electron diffraction spectroscopy (EDS). Porosity and void volume analysis were carried out to characterize the matrices. Chondrocytes were isolated from the proximal humerus of 22 week-old male, adult, local albino rabbits. For cell type characterization, Type II collagen was measured by Western Blot analysis. The foams were seeded with 1×10 6 chondrocytes and histological examinations were carried out to assess cell–matrix interaction. Macroscopic examination showed that PHBV (with or without chondrocytes) maintained its integrity for 21 days, while CaP–Gelfix ® was deformed and degraded within 15 days. Cell-containing and cell-free matrices were implanted into full thickness cartilage defects (4.5 mm in diameter and 4 mm in depth) at the patellar groove on the right and left knees of eight rabbits, respectively. In vivo results at 8 and 20 weeks with chondrocyte seeded PHBV matrices presented early cartilage formation resembling normal articular cartilage and revealed minimal foreign body reaction. In CaP–Gelfix ® matrices, fibrocartilage formation and bone invasion was noted in 20 weeks. Cells maintained their phenotype in both matrices. PHBV had better healing response than CaP–Gelfix ®. Both matrices were effective in cartilage regeneration. These matrices have great potential for use in the repair of joint cartilage defects.

The biomechanical environment of a bone fracture and its influence upon the morphology of healing

The mechanism by which mechanical stimulus of reparative tissues directs the pattern of healing of a bone fracture is not understood. Several hypotheses have been developed that predict the ossification pattern during healing for a given ambient mechanical environment. These have remained unproved because of the absence of data on stress fields in the reparative tissue of real fractures. The present study examines the predictive performance of the most recent hypothesis that was proposed by Claes and Heigele (J. Biomech. 32 (1999) 255), against measured and calculated data from the clinical fracture reported by Gardner et al. (J. Biomech. 33 (2000) 415). The hypothesis was used to predict ossification from the preceding stress and strain fields present in the FEM of Gardner et al. at four temporal stages during healing. Predictions were then compared with the observed differentiation and maturation. During early healing of the interfragmentary gap region, the hypothesis correctly predicted the formation of connective tissue and fibrocartilage, and during later healing it correctly predicted the beginning of endochondral ossification. At the periphery of the periosteal callus, the hypothesis correctly predicted intramembraneous ossification during early healing, and also its thickening by endochondral ossification during later healing. However, the hypothesis incorrectly predicted intramembraneous ossification during early healing of the main periosteal callus, although in later healing it correctly predicted endochondral ossification.

Enveloping the tendon graft with periosteum to enhance tendon-bone healing in a bone tunnel: A biomechanical and histologic study in rabbits

Purpose: Fixing and incorporating the tendon graft within the bone tunnel is a major concern when using grafts for ligament reconstruction. The periosteum contains multipotent stem cells and has the potential to form osteogenic and chondrogenic tissues. This study uses histologic and biomechanical analyses to examine the effect of periosteum on tendon-bone healing within a bone tunnel. Type of Study: Experimental study in an animal model. Methods: In this study, 36 adult New Zealand White rabbits were used. The long digitorum extensor tendon was transplanted into a bone tunnel of the proximal tibia. The periosteum from the proximal tibia was sutured on the surface of the tendon portion. The tendon was pulled through a drill-hole in the proximal tibia and attached to the medial aspect of the tibia. Histologic examination of the tendon-bone interface and biomechanical test for maximal pullout load were evaluated at 4, 8, and 12 weeks after operation. Results: Histologic analysis of the tendon-bone interface showed a fibrous layer formed between the tendon and the bone by the periosteum. This layer became progressively integrated with the tendon and bone surface during the healing process. At 4 weeks, the cancellous bone lining in the bone tunnel was interdigitated with the fibrous interface tissue. At 8 weeks, progressive new bone grew into the interface fibrous layer. At 12 weeks, collagen fibers anchored to the bone and organization with fibrocartilage formation developed between the tendon and bone. Biomechanical testing revealed higher maximal pullout strength in the periosteum-enveloped group at all time points, with a statistically significant difference at 8 and 12 weeks. The periosteum-treated group had a higher interface strength-to-length ratio and significant increase at 8 weeks and 12 weeks. Conclusions: The histologic and biomechanical studies demonstrated that, if periosteum was sutured on the tendon that was transplanted within a bone tunnel, it resulted in a superior healing process and better healed strength. When doing ligament reconstruction with a tendon graft, the periosteum can be sutured to the graft to enhance tendon-bone healing.Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 19, No 3 (March), 2003: pp 290–296

Arthroscopic techniques for cartilage repair

Abstract Joint injuries are a major concern for horses used in athletic and recreational sports. Spontaneous cartilage repair diminishes as horses reach maturity, and surgical measures have been developed to bolster these meager intrinsic responses. Local debridement, joint lavage, and marrow stimulation techniques provide improved symptomatic therapy that may last several years. Subchondral drilling (forage) has largely been superseded by microfracture techniques to open the subchondral bone in damaged cartilage areas. Marrow-derived cells and growth factors then contribute to better fibrocartilage formation. Transplantation techniques can result in more durable hyaline cartilage but add a level of complexity to cartilage repair that can be costly, time-consuming, and occasionally painful. Autogenous osteochondral dowel transplantation or mosaicplasty provides an integrated weight-bearing island that may sustain better loads than cell-grafting methods. However, donor-site morbidity and technical difficulties both in harvest and implant of osteochondral grafts dampens the promise of major hyaline cartilage rejuvenation. Equine joint repair demands arthroscopic methods, and chondrocyte and pluripotent stem-cell implantation provide promise of improved cartilage repair. Combination of insulin-like growth factor-I and chondrocytes provides a clinically useful grafting method for extensive osteochondritis dissecans (OCD), subchondral cysts, and traumatic injuries. Future directions look toward gene-enhanced cell transplants that may provide a major early matrix response and durable in situ cartilage reformation.

Surgical approaches to the repair of cartilage defects

Traumatic defects in articular cartilage are common in the young athletic population. They may occur in association with other injuries to the knee, particularly anterior cruciate ligament disruption. We understand that both theoretically and clinically, these defects may lead to an increased incidence of ongoing painful symptoms in the short term and degenerative joint disease in the long term. The orthopedic surgeon has several options to deal with theses injuries. The choice is dependent on the size and position of the lesion in the knee and any associated pathology. The most commonly practised methods aim to evoke a ‘repair’ response of fibrocartilage formation. This often helps in the short term, but the fibrocartilage degenerates over time, resulting in the return of symptoms. These ‘repair’ methods can be classified as: 1. Marrow stimulation techniques– these include abrasion chondroplasty, subchondral drilling and microfracture techniques. 2. Scaffold induced repair techniques. More recently, attempts to ‘re-generate’ normal articular cartilage have been introduced to clinical practice. These are: 1. Mosaicplasty– autogenous osteochondral plug transplantation. 2. Autologous chondrocyte implantation (ACI)– this involves the harvest and culturing of autologous chondrocytes and then re-implantation via injection under a periosteal or porcine collagen patch. The newest technology allow implantation of the collagen patch which has had chondrocytes cultured within its structure. The results of these autologous chondrocyte implantation show encouraging short to medium term results clinically. 3. Some orthopaedic centres have recently been advocating ‘articular cartilage paste grafting’– this is a simple technique based upon the theory that a paste of autogenous articular cartilage and subchondral bone should contain chondrocytes, stem cells and growth factors in its own scaffold. Orthopedic surgeons await with interest the outcome of pre-clinical work in examining the use of growth factors, scaffold technology and gene therapy in helping with the clinical problem of articular cartilage defects.

Mechanobiology of initial pseudarthrosis formation with oblique fractures

Mechanical stresses play an important role in regulating tissue differentiation in a variety of contexts during skeletal development and regeneration. It has been shown that some intermittent loading at a fracture site can accelerate secondary fracture healing. However, it has not been shown how the stress and strain histories resulting from mechanical loading of a fracture might, in some cases, inhibit normal fracture healing and induce pseudarthrosis formation. In this study, finite element analysis is used to calculate hydrostatic stress and maximum principal tensile strain patterns in regenerating tissue around the site of an oblique fracture. Using a mechanobiologic view on tissue differentiation, we compared calculated stress and strain patterns within the fracture callus to the histomorphology of a typical oblique pseudarthrosis. Tissue differentiation predictions were consistent with the characteristic histomorphology of oblique pseudarthrosis: in the interfragmentary gap, tensile strains led to “cleavage” of the callus; at the ends of both fracture fragments, hydrostatic pressure and tensile strain caused fibrocartilage formation; and, at discrete locations of the periosteum at the oblique fracture ends, mild hydrostatic tension caused bone formation. We also found that discrete regions of high hydrostatic pressure correlated with locations of periosteal bone resorption. When previous findings with distraction osteogenesis are considered with these observations, it appears that low levels of hydrostatic pressure may be conducive to periosteal cartilage formation but high hydrostatic pressure may induce periosteal bone resorption during bone healing. We concluded that tissue differentiation in pseudarthrosis formation is consistent with concepts previously presented for understanding fracture healing, distraction osteogenesis, and joint formation.

Regional adaptations in three rat tendons

Although detailed histological and immunocytochemical studies have been published for the rat calcanear tendon (CT), little is known of the structure, composition and biomechanics of the deep (DFT) and superficial (SFT) flexor tendons. In this study, we examined the structural specialization of these three tendons in 90-day-old rats by applying histochemical and biochemical assays to different tendon regions (proximal, intermediate and distal regions of the DFT and SFT, and proximal and distal regions of the CT). There were regional differences in tissue structure, glycosaminoglycan type and content, swelling properties and in the amount and distribution of elastic fibers. Dermatan sulfate occurred in all regions, but chondroitin sulfate predominated in the intermediate region of the DFT and in the distal region of the CT. These two chondroitin sulfate-bearing regions showed swelling in water, while all other regions lost fluid in water. Fibrocartilaginous sites were observed on the CT, one at the insertion to the bone and another distally at the innermost area of the tendon. The intermediate region of the DFT showed round cells disposed in lacunae, while the proximal and distal regions were typically fibrous. The intermediate region of the SFT showed a wavy array of collagen bundles but neither toluidine blue staining in the matrix nor round cells. Elastic fibers were present in each region of the three tendons, but were more prominent in the intermediate zone of the SFT. These results demonstrate regional variation in the three tendons. Tendon differentiation may occur by an increase in the number of elastic fibers and by variations in the arrangement of collagen fibers, without fibrocartilage formation.