Differential metabolic responses of periarticular ligaments and tendon to joint immobilization.

Parameters of collagen metabolic behavior were analyzed in the periarticular connective tissues, i.e., medial collateral ligament (MCL), anterior cruciate ligament (ACL), and patellar tendon (PT), of control and immobilized rabbit knees. Two periods of immobilization were studied: 9 and 12 wk. Collagen turnover and collagen cross-links were quantitatively assessed in the three tissues. The results showed that after 9 wk both synthesis and degradation were significantly increased in the MCL and ACL, whereas the PT showed lesser effects. After 12 wk all three tissues experienced significant losses of collagen mass, which resulted in tissue atrophy. The concentrations of the reducible collagen cross-links dihydroxylysinonorleucine and hydroxylysinonorleucine in the immobilized MCL and ACL were greater than their respective controls, indicating an increase in collagen synthesis, whereas concentrations of the nonreducible cross-link hydroxypyridinoline were observed to be decreased in these tissues. Of the reducible cross-links in the PT, only hydroxylysinonorleucine was found to be increased over control, whereas hydroxypyridinoline was slightly less concentrated. These results taken together have demonstrated that the ligamentous tissues are more susceptible to the effects of stress deprivation secondary to joint immobilization than the PT, and, in particular, the ACL of the three tissues studied appears to be most vulnerable.

Collagenase activity in anterior cruciate ligament: protective role of the synovial sheath.

To evaluate the protective role of the synovial sheath of the anterior cruciate ligament (ACL), we have developed a synovectomy model that exposes the ACL substance to the intra-articular environment with and without hemarthrosis. Histology and the level of collagenase activity were studied to assess intrinsic ligament alterations. The treatment groups studied were as follows: ACLs of sham-operated knees receiving arthrotomy only, ACLs of knees receiving arthrotomy and acute hemarthrosis, ACLs of knees that underwent synovectomy, and ACLs of knees that underwent both synovectomy and acute hemarthrosis. All animals were killed 10 days postoperatively for gross, histological, and biochemical assessment. Histologically at 10 days ACLs experiencing synovectomy and ACLs having synovectomy plus hemarthrosis revealed marked hypocellular areas. Biochemical results indicate that synovectomy is the treatment mainly responsible for the observed increase in ACL collagenase activity. Hemarthrosis alone clearly had no effect, although hemarthrosis coupled with synovectomy appeared to further increase the amount of active collagenase present in the ACLs. This study indicates that, with exposure of the ACL substance to the synovial fluid or with hemarthrosis after synovectomy, there is an increase in the degradative activity of the ACL. The protective role of the synovial sheath suggests that the synovial sheath injury associated with acute ACL rupture may allow for exposure of the ligament substance to the degradative effects of the synovial environment and associated hemarthrosis.

Medical collateral ligament healing subsequent to different treatment regimens.

The response of transected canine medical collateral ligaments (MCL) to clinical treatment regimens was investigated. These regimens included no surgical repair with no immobilization and surgical repair with various periods of immobilization. The biomechanical, biochemical, and histological properties of the healing MCL were examined 6 and 12 wk postoperatively. At 6 wk, all healing MCLs had increased cellularity with decreased levels of total collagen and increased amounts of reducible Schiff base cross-links and type III collagen. Biomechanically, the varus-valgus (V-V) knee laxity was significantly increased, and no group achieved normal structural or mechanical properties. At 12 wk the histological appearance of the MCL became more normal but still had increased cellularity. Biochemically, the total collagen levels in experimental MCLs were not statistically different from the controls, but these MCLs still had high amounts of type III collagen and an even higher number of reducible cross-links. From knees in which the MCL was not treated, the V-V knee laxity and the ultimate loads of the femur-MCL-tibia complex achieved normal values. However, the stress-strain properties for these MCLs and those treated with repair and immobilization did not completely recover.

Early histologic, metabolic, and vascular assessment of anterior cruciate ligament autografts.

A rabbit model for anterior cruciate ligament (ACL) reconstruction using autogenous patellar tendon was utilized to study the early events of autograft cellular dynamics. Biochemical, autoradiographic, histological, and vascular injection techniques demonstrated that the native autograft cell population rapidly necroses. This repopulation occurs without a vascular contribution; cells entering the autograft are reliant upon synovial fluid nutrition.

Autogenous intrasynovial and extrasynovial tendon grafts: an experimental study of pro alpha 1(I) collagen mRNA expression in dogs.

On the basis of recent evidence that the healing processes of tendon grafts are donor-tissue specific, in situ hybridization, using a 372 bp cDNA fragment complementary to a portion of pro alpha 1(I) collagen mRNA, was utilized to compare the cellular responses to transplantation exhibited by autogenous intrasynovial and extrasynovial flexor tendon grafts. Intrasynovial and extrasynovial tendons from the hindpaw were transferred to synovial sheaths in the forepaw of 12 mongrel dogs (24 tendons) and treated with immediate controlled passive motion. The tendon grafts were harvested at 2, 4, and 6 weeks, and each was divided into a proximal, central (8 mm), and distal portion. Sections from the central portion were embedded in paraffin and subjected to in situ hybridization, autoradiography, and staining; levels of procollagen mRNA then were assessed by microscopic examination. The two types of tendon grafts exhibited different levels of pro alpha 1(I) collagen mRNA expression at all three time points. Intrasynovial tendon grafts displayed no areas of increased type-I procollagen mRNA at 2, 4, and 6 weeks. The extrasynovial tendon grafts displayed increased surface levels of type-I procollagen mRNA at 2 and 4 weeks; the levels decreased to background levels by 6 weeks. The high levels of procollagen mRNA exhibited by the extrasynovial grafts suggest increased collagen synthetic activity, indicative of a cellular response to injury, whereas the preservation of low levels of expression in the intrasynovial grafts may signify a less inflammatory cellular response.

The phenomenon of "ligamentization": anterior cruciate ligament reconstruction with autogenous patellar tendon.

Reconstruction of the anterior cruciate ligament (ACL) with patellar tendon (PT) is a common procedure for the symptomatic ACL-deficient knee. Questions regarding graft incorporation, viability, and nutrition of the transplanted tissue are of concern. This relates to the graft's response to its new intrasynovial milieu and new physical forces. These factors were studied in a rabbit model of ACL reconstruction using PT and were evaluated with histological and biochemical parameters with respect to time. A histological and biochemical metamorphosis of the grafted PT occurred in this study. Autografts demonstrated a gradual assumption of the microscopic properties of normal ACL; by 30 weeks postoperatively, cell morphology was ligamentous in appearance. Normally, type III collagen is not observed in PT, however, a gradual increase in its concentration was seen in the grafts; by 30 weeks its concentration (10%) was the same as in normal ACL. Similarly, glycosaminoglycans content increased from its normally low level in PT to that found in native ACL. Collagen-reducible crosslink analysis demonstrated that grafted tissue changed from the normal PT pattern of low dihydroxylysinonorleucine (DHLNL) and high histidinohydroxymerodesmosine (HHMD) to the pattern seen in normal ACL (high DHLNL and low HHMD) by 30 weeks. These data suggest that when PT is placed in the anatomic and environmental milieu of the ACL, a "ligamentization" of the grafted tissue results; also the autograft initially depends on synovial fluid nutrition, as revascularization occurs after 6 weeks.

Chondrogenic phenotype of perichondrium-derived chondroprogenitor cells is influenced by transforming growth factor-beta 1.

Our laboratory has developed a method for the repair of osteochondral defects by implanting cultured perichondrial cells attached to a biodegradable polylactic acid scaffold. The success of this approach depends in part on the proliferative characteristics and the phenotype of the implanted cells. Transforming growth factor-beta 1 has been reported to influence these parameters in several mesenchymal-derived tissues in vitro and in vivo. The chondrocytic phenotype is marked by an enhanced expression of the collagen type-II gene. In this study, cultures grown from explants of rabbit rib perichondrium were exposed to exogenously added transforming growth factor-beta 1 at concentrations of 0.1-10 ng/ml of media. Cell proliferation and collagen gene expression were measured. The expression of types I and II collagen genes was analyzed by Northern blot and reverse transcriptase-polymerase chain reaction. The exogenous addition of transforming growth factor-beta 1 at a concentration of 0.1-10 ng/ml resulted in tritiated thymidine uptake by perichondrial cells, with optimum proliferative effects at 0.1 ng/ml. Transforming growth factor-beta 1 added at concentrations of 0.1 and 0.5 ng/ml significantly upregulated the expression of type-II collagen mRNAs. The results suggest that, when the chondrocytic phenotype is defined by markedly enhanced type-II collagen gene expression, the chondrocytic phenotype of explant cultures of perichondrium-derived cells is enhanced by the exogenous addition of transforming growth factor-beta 1.

The early effect of high molecular weight hyaluronan (hyaluronic acid) on anterior cruciate ligament healing: an experimental study in rabbits.

The purpose of this study was to assess, morphologically and biochemically, the effect of hyaluronan (HA) on the early repair process of the anterior cruciate ligament (ACL). Following partial bilateral laceration in the midsubstance of the cruciate ligament, a single dose of HA (MW of 3.6 x 10(6] was injected in one knee and saline in the contralateral knee. Postsurgery, the rabbits were allowed normal (nonimmobilized) cage activity, and were killed after 4 (n = 11) and 12 (n = 10) weeks. The ligaments were evaluated by gross morphology and graded according to the degree of repair. We used grades 1,2, and 3 for uncovered, partially covered, and totally covered lacerations, respectively. Five of the HA-treated ligaments at each time studied were completely covered, compared to 0 at 4 weeks, and 1 at 12 weeks in the saline group. Paired evaluations of the lacerated ACLs showed that the HA-treated ligaments received a healing grade higher than the ligaments exposed to saline in 14 of the 21 animals. In the remaining animals, there was no difference between the sides. The repaired tissue of the ACLs was also examined by light and electron microscopy. When compared qualitatively with saline controls, HA-treated ligaments exhibited a more pronounced repair, with an increased angiogenesis and less inflammatory response. Biochemical analysis demonstrated a mean higher value of type III collagen in the HA-treated injured ACL than in saline-treated injured ACL (13.4 +/- 1.1% and 11.0 +/- 0.8%, respectively). This increased synthesis of type III collagen in the HA-treated injured ACL was statistically higher (p less than 0.05) when compared to the saline-treated injured ACL.

Injury of the anterior cruciate ligament: the role of collagenase in ligament degeneration.

Rapid degeneration of the anterior cruciate ligament (ACL) has been observed following acute ACL rupture. An understanding of this process might explain some of the poor clinical results of primary ACL repair. We created a surgical rabbit model of acute ACL injury and developed an in vitro assay for collagenase activity in the ACL and menisci. Microscopic evaluation revealed a rapidly degenerative process in injured ACLs, with loss of cellularity and matrix organization. This was associated with a significant increase in collagenase activity and a decrease in total collagen of the injured ACLs as compared with sham-operated controls. These findings confirm the observation that cut ACL ligament ends rapidly degenerate. This degenerative process might be partly due to a response of cells intrinsic to the ACL to injury. Left unchecked, this process may be detrimental to surgical attempts for primary ACL repair.

Characterization of arthroplasty tissue after 14 years post-cup arthroplasty: a morphological and biochemical assessment.

The cup arthroplasty has been reported to cause the formation of a fibrocartilaginous joint surface, which may result in a painless, functional joint. The joint surface of a 38-year-old man with a failed cup arthroplasty implanted for 14 years was examined histologically and biochemically. The joint surface tissue of this patient resembled fibrous connective tissue, with major types of collagen being Type I and Type III. No evidence of cartilaginous transformation in the healing scar was demonstrated, despite several years of successful functioning of the cup arthroplasty.

Chondrocyte apoptosis and regional differential expression of nitric oxide in the medial meniscus following partial meniscectomy.

Partial medial meniscectomy leads to tibial articular cartilage degeneration. Nitric oxide (NO) production increases with the development of osteoarthritis (OA) and has been shown to have a catabolic effect on chondrocytes. Since distribution of chondrocytic and fibroblastic cell types within the total cell population comprising meniscus is region-specific, we compared NO production in the peripheral and central regions of the medial meniscus 12 weeks after partial medial meniscectomy and assessed chondrocyte apoptosis and NO production in the tibial articular cartilage. Additionally, transcriptional gene expression of inducible nitric oxide synthetase (iNOS) and immunohistochemical staining of nitrotyrosine were examined. The results showed that following partial medial meniscectomy, NO production in the central region of the medial meniscus and in the tibial articular cartilage were significantly higher than respective NO levels in normal and sham-operated controls. Reverse transcription polymerase chain reaction (RT-PCR) revealed a high transcriptional expression of the iNOS gene in the central region of the meniscus and in tibial articular cartilage following partial medial meniscectomy. Nitrotyrosine immunoreactivity was prominent in the central region of the medial meniscus and in the deep layer of the tibial articular cartilage and apoptotic cells were also detected in situ in the superficial zone of the tibial articular cartilage and central regions of the medial meniscus following partial medial meniscectomy. These observations suggest that the central region of the meniscus is responsible for NO synthesis associated with apoptosis in both meniscal and articular cartilage cells following partial meniscectomy.

The effects of hyaluronan on the meniscus and on the articular cartilage after partial meniscectomy.

The effect of hyaluronan (molecular weight = 8 x 10(5)) on the meniscus and on the articular cartilage was assessed after partial meniscectomy in a rabbit model. On gross examination, remodeled meniscus appeared as newly synthesized translucent tissue, and was seen in both vehicle- and hyaluronan-treated menisci. Histologically, safranin O staining revealed the strong presence of glycosaminoglycans in the newly remodeled tissue, and polarized light demonstrated the absence of mature collagen architecture. Hydration of the hyaluronan-treated menisci was significantly less than that of the vehicle-treated menisci, and the reducible collagen cross-link dihydroxylysinonorleucine was significantly increased in the hyaluronan-treated menisci compared with the vehicle-treated menisci, indicative of a greater degree of collagen remodeling. In situ hybridization of vehicle- and hyaluronan-treated menisci revealed a high level of type I procollagen mRNA expression and minor expressions of types II and III mRNA. Expression of the type I collagen gene appeared to be more pronounced in the hyaluronan-treated menisci than in the vehicle-treated menisci. The tibial plateaus revealed mild cartilage fibrillation after partial meniscectomy. A statistically significant difference between vehicle- and hyaluronan-treated cartilage was not demonstrated in the present study because of the slow development (i.e., 12 weeks) of osteoarthritis after partial meniscectomy in the rabbit model. These results suggest that in the rabbit model, hyaluronan enhances collagen remodeling and inhibits meniscal swelling after partial meniscectomy in the avascular region.

The effects of hyaluronan on tissue healing after meniscus injury and repair in a rabbit model.

To assess the effect of hyaluronan on meniscus injury and repair, we had 35 mature New Zealand White rabbits undergo bilateral meniscus injury and repair (19 in the peripheral region, and 16 in the inner region). A longitudinal tear was created in the medial meniscus and repaired with horizontally placed nylon sutures. The left knee joint received intraarticular injections of hyaluronan 1 week after surgery and once a week for 5 weeks. The right knees were injected with phosphate-buffered saline (the carrier vehicle of the hyaluronan). Twelve weeks after repair, tears in the peripheral region showed gross and histologic evidence of healing, with no difference between the vehicle- and hyaluronan-treated menisci. Biochemically, the ratio of reducible collagen cross-links in the hyaluronan-treated menisci was significantly higher than in the vehicle-treated menisci, indicating greater level of collagen remodeling. Biomechanically the vehicle- and hyaluronan-treated menisci demonstrated similarly high tearing load and fracture toughness. In the inner region, poor healing response was observed grossly and histologically in both treatment groups. Water content in the hyaluronan-treated menisci was significantly lower than in the vehicle-treated menisci, indicating a lower level of swelling. Hyaluronan treatment stimulated collagen remodeling in the peripheral region and inhibited swelling of the meniscus repaired in the inner region.

Long-term fate and effects of exercise on sternal cartilage autografts used for repair of large osteochondral defects in horses.

Bilateral osteochondral defects (10 mm2 x 3 mm deep) were created on the distal articular surface of the radial carpal bone of ten, 2- to 3-year-old horses. One defect of each horse was repaired, using a sternal cartilage autograft (treated), and the other was left untreated (control). The horses were exercised on a high-speed treadmill at incrementally increased speed and duration over the course of 12 months. Horses were evaluated arthroscopically at 6 to 7 weeks, and clinical examinations were conducted weekly at exercise. Twelve months after surgery, carpuses of each horse were radiographed and clinically examined prior to euthanasia. A gross pathologic evaluation of each joint was conducted, and samples were collected for histologic, histochemical, histomorphometric, and biochemical evaluation. Radiographically, the grafted joints had more extensive evidence of arthropathy, and clinically, 8 of the 10 horses were more lame in the grafted limb. On the basis of histomorphometry, the repair tissue of the grafted defects contained a greater median percentage of hyaline cartilage (45%) than that of control defects (4.5%), and the control defects contained a greater percentage of fibrocartilage (82%) than did grafted defects (28.5%). A greater median percentage of repair tissue stained with safranin-O in the grafted defects (24.5%) than in the control defects (3.5%). On gross pathologic and histologic evaluation, repair tissue of the control defects had better continuity and was more firmly attached to the subchondral bone than was repair tissue of the grafted defects. Repair tissue of the grafted defects had extensive fissure and flap formation. Histologically, subchondral bone reactivity and fibroplasia was extensive in grafted joints. Repair tissue of grafted defects had a greater percentage of type II collagen (mean +/- SEM, 83.5 +/- 2.95%) than did controls (mean, 79.4 +/- 3.87%) that was not statistically significant. Hexosamine content was significantly higher (P < 0.05) in repair tissue of the grafted defect (mean, 28.9 +/- 3.00 mg/g of dry weight) vs control (mean, 20.6 +/- 1.85 mg/g of dry weight). On the basis of this experimental model, sternal cartilage autografts cannot be recommended at this time for repair of osteochondral defects in athletic horses.

The effects of immobilization on the maturation of the anterior cruciate ligament of the rabbit knee.

Immobilization-induced alterations occurred in young anterior cruciate ligament (ACL) samples, including the loss of the rounded appearance of the cells. The mature ACL was minimally altered by immobilization at the light microscopy level. In the immobilized young ACL the fibroblasts became elongated and there was loss of the normal pericellular matrix. The immobilized mature ACL differed from controls primarily in the intracellular composition, as there was significantly more rough endoplasmic reticulum (RER) present. Collagen concentrations were reduced only in young immobilized ACL, while no differences were observed in the mature ACL. The collagen synthesis rate in the mature ACL increased with immobilization, although no significant change was observed in the young ACL. The increase in the rate of synthesis of the stress deprived ACL in the mature animals reflected an increase in collagen turnover rather than an increase in accumulation of collagen.

Growth factor expression in healing rabbit medial collateral and anterior cruciate ligaments.

Exogenously administered growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta) and basic fibroblast growth factor (bFGF) have been shown to affect connective tissue healing in vivo, but their intrinsic role in the healing response has not been established. In the present study, immunohistochemistry with antibodies directed against these growth factors showed that expression of PDGF, TGF-beta 1 and bFGF was increased in and around the wound site in the rabbit medial collateral ligament (MCL) seven days following surgical injury. The strong expression of PDGF correlated with the observed increased cellularity consistent with this growth factor's mitogenic and chemotactic properties. Expression of these growth factors was also increased in wounded rabbit anterior cruciate ligaments (ACL) at seven days following surgical injury, but such expression was limited to the edge of the ACL injury site and was of lesser intensity relative to the MCL. This study suggests that PDGF and TGF-beta 1, and to a lesser extent bFGF, are actively involved during the early stage of MCL healing, but have a more limited presence in the injured rabbit ACL.

Decrease in fibronectin occurs coincident with the increased expression of its integrin receptor alpha5beta1 in stress-deprived ligaments.

Stress deprivation secondary to immobilization leads to atrophic changes in periarticular soft tissues. The changes in ligaments include a disorganization of collagen and cellular ultrastructure with varied biochemical alterations resulting in a functionally weaker tissue. This study tests the hypothesis that alterations in fibronectin (Fn) and the expression of its integrin receptor alpha5beta1 in ligament fibroblasts accompany the extracellular matrix remodeling which occurs in stress-deprived knee ligaments. The left knees of eighteen New Zealand white rabbits were surgically immobilized in acute flexion. Fibroblasts within three nine week and three twelve week stress-deprived anterior cruciate ligaments (ACLs) and medial collateral ligaments (MCLs) demonstrated markedly increased immunostaining for the beta1 and alpha5 integrin subunits, as compared to fibroblasts in the contralateral unoperated control ligaments. The effects of stress deprivation on the concentration of Fn was measured by competitive ELISA on the remaining twelve rabbits. Decreases in Fn of 54.0 percent and 63.7 percent occurred in the ACL after nine and twelve weeks of stress deprivation when compared to contralateral controls. The MCL had less of a decrease, losing 37.7 percent and 41.7 percent at nine and twelve weeks, respectively. These results suggest an important role for the Fn-specific integrin receptor alpha5beta1 in remodeling stress-deprived periarticular ligamentous tissue, and the importance of maintaining normal stresses on periarticular ligaments to prevent the degradation of extracellular matrix components such as Fn.

Transforming growth factor beta one (TGF-beta 1) enhancement of the chondrocytic phenotype in aged perichondrial cells: an in vitro study.

BACKGROUND: Perichondrium is recognized as a tissue with chondrogenic potential yielding cells which can be used for osteochondral repair. Factors which influence the proliferative ability and chondrocytic phenotype of such cells include age and presence of specific growth factors, i.e. TGF-beta 1. The present in vitro study assessed proliferation and markers of chondrocytic phenotype in cells extracted from the rib perichondrium of four- to five-year-old aged rabbits, and assessed the effects of exogenously added TGF-beta 1 on those cells. METHODS: Assays included 3H-thymidine incorporation (cell proliferation), 35S-sulfate incorporation (proteoglycan synthesis) and quantitative RT-PCR for determination of type II collagen gene expression. RESULTS: The results demonstrated that addition of TGF-beta 1 to the culture media stimulated thymidine incorporation and proteoglycan synthesis up to four- and five-fold, respectively, in aged perichondrium-derived cells. Moreover, the exogenous addition of TGF-beta 1 to the culture media resulted in an upregulation of transcriptional expression of the type II collagen gene. CONCLUSIONS: In summary, the present study has demonstrated that exogenously added TGF-beta 1 can stimulate proliferation and chondrocytic phenotype in aged perichondrium-derived cells in vitro.

Characterization of pro-apoptotic and matrix-degradative gene expression following induction of osteoarthritis in mature and aged rabbits.

OBJECTIVE: The genetic and molecular changes leading to the distinctive alterations of aged cartilage and its propensity for developing osteoarthritis (OA) are unknown. We hypothesized that pro-apoptotic and matrix-degradative gene expression in a rabbit model of induced OA using mature and aged animals might elucidate this relationship. METHODS: Groups of six mature and aged rabbits underwent anterior cruciate ligament transection (ACLT) and were sacrificed 4 weeks after surgery to create an Outerbridge grade II OA. RNA was extracted from the articular cartilage and menisci of the affected knee and was examined with regard to expression of the following genes: Caspase 8, Fas, Fas ligand (Fas-L), p53, aggrecanase, matrix metalloproteinase (MMP)-1, and MMP-3-MMP-13. A second cohort of mature and aged animals was sacrificed with no intervention to the joint and gene expression was assessed in a similar manner. RESULTS: Fas and Caspase 8 showed significantly increased expression in the cartilage of mature animals with induced OA when compared to unoperated controls while induction of OA in aged rabbits did not significantly increase expression of any of the apoptosis genes. Among unoperated animals, the aged cohort showed significantly increased expression of MMP-1 and aggrecanase in cartilage when compared to mature animals. MMP-13 expression was upregulated in aged cartilage following induction of OA. Although ACLT animals showed gross thinning and irregularities within the meniscus, only the expression of Caspase 8 in the aged rabbits was significantly increased after induction of OA. CONCLUSIONS: Aging of articular cartilage shares some qualities with the development of OA, as seen in the parallel increases in gene expression of Caspase 8 and Fas. Although this may imply a common mechanism of cartilage degeneration in aging and OA or even a spectrum of disease, both are complex processes requiring further study.

Age-related properties of medial collateral ligament and anterior cruciate ligament: a morphologic and collagen maturation study in the rabbit.

The medial collateral ligament (MCL) and anterior cruciate ligament (ACL) from New Zealand white rabbits ages 2, 12, and 36 mos were resected and utilized for analysis by light microscopy and electron microscopy, as well as for determinations of water content, collagen concentration, collagen crosslink (i.e., reducible, nonreducible) analysis, and collagen synthesis rates in vitro. Both modes of microscopy revealed substantial differences between the various age groups. Water content, as well as collagen concentration, decreased significantly from the 2-mo to the 12- or 36- mo groups. The concentration of the reducible crosslinks, associated with less mature tissue, was significantly greater in the younger tissues relative to the aged tissues. The nonreducible crosslink increased with maturation of the collagen in the 36-mo rabbits. Collagen synthesis rates fell from a highest value in the 2-mo group to the lowest in the 36-mo group.