Differences in collagen metabolism between normal and osteoarthritic human articular cartilage.

Normal human articular cartilage synthesizes only one type of a chain, which exhibits the chromatographic behavior of the alpha(l)(II) chains described for chick and bovine cartilage. Osteoarthritic cartilage, on the other hand, synthesizes in addition a collagen containing alpha(2) chains and beta components. The different structural features of the two types of collagen may account for some of the functional defects of osteoarthritic cartilage.

Calcitonin stimulates bone formation when administered prior to initiation of osteogenesis.

The influence of calcitonin (CT) on various stages of bone formation was investigated. A demineralized collagenous bone matrix-induced bone forming system in rats was used to temporally segregate chondrogenesis and osteogenesis. Administration of CT (15 Medical Research Council Units [MRCU]) daily) at the initiation of matrix-induced bone formation (BF) resulted in a 76% stimulation of BF as measured by 45Ca incorporation and alkaline phosphatase activity. This increase was due, in part, to a stimulation of cartilage and bone precursor cell proliferation monitored by the rate of [3H]thymidine incorporation and ornithine decarboxylase activity. Chondrogenesis on day 7 as measured by 35SO4 incorporation was increased by 52% with CT treatment. To rule out the possibility of a secondary response due to parathyroid hormone, similar studies were done in parathyroidectomized animals and CT stimulation of BF was still observed. However, when CT injections were started after cartilage formation (day 8) there was no stimulation of BF but a significant decrease in 45Ca incorporation was observed. These results indicate CT has two actions: (a) when CT is administered during the initial phases of bone formation, it increases BF due to a stimulation of proliferation of cartilage and bone precursor cells; and (b) when CT is administered after bone formation has been initiated, subsequent bone formation is suppressed.

Abnormalities in the biosynthesis of cartilage and bone proteoglycans in experimental diabetes.

Proteoglycans synthesized in developing cartilage and bone were investigated in control and streptozotocin-induced (65 mg/kg, i.v.) diabetic rats. Ten days after streptozotocin injection, animals were implanted subcutaneously with demineralized bone matrix particles. This system induces formation of cartilage and bone on days 7 and 14, respectively. Two hours before they were killed, animals were injected with 35SO4 and the labeled proteoglycans were extracted from the explants and metaphyses by either a direct associative extraction (0.5 M GuCl2) or a direct dissociative extraction (4.0 M GuCl2). These procedures extract 80-90% of the total counts incorporated. To characterize the proteoglycans, extracts were subjected to cesium chloride density gradient centrifugation and molecular sieve chromatography. These data showed that (1) there is less proteoglycan made in diabetic bone; (2) the proteoglycan aggregate is of a smaller molecular weight in bone than in cartilage; (3) 10% of the proteoglycan synthesized in diabetic bone was in the form of aggregates compared with 48% of the control bone; (4) aggregates did form in the diabetic cartilage, and their molecular weight was smaller than in normal cartilage. This investigation shows that proteoglycans, structurally important macromolecules of cartilage and bone, are altered in experimental diabetes. This metabolic abnormality may be an important factor contributing to decreased bone formation observed in diabetes.

Selection and amplification of a bone marrow cell population and its induction to the chondro-osteogenic lineage by rhOP-1: an in vitro and in vivo study.

The differentiation and maturation of osteoprogenitor cells into osteoblasts are processes which are thought to be modulated by transforming growth factors-beta (TGF-beta) as well as by bone morphogenetic proteins (BMPs). Osteogenic protein-1 (OP-1, also known as BMP-7) is a member of the BMP family, and it is considered to have important regulatory roles in skeletal embryogenesis and bone healing. Rat bone marrow cells were cultured in vitro in a collagen-gel medium containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of 40 ng/ml recombinant human OP-1 (rhOP-1). Under these conditions, survival of the bone marrow cell population was dependent on the presence of rhOP-1. Subsequently, the selected cells were cultured-for 6 days in medium containing 40 ng rhOP-1 and 10% FBS. During the last 2 days, dexamethasone (10(-8) M) and beta-glycerophosphate (2 mM) were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels, colony number and size were determined. Chondro-osteogenic differentiation in vitro was evaluated in terms of the expression of alkaline phosphatase, the production of osteocalcin and the formation of mineralized matrix. After culturing in vitro, cells were placed inside diffusion chambers or inactivated demineralized bone matrix (DBM) cylinders and implanted subdermically into the backs of old rats for 28 days. Biochemical, histological and immunocytochemical analyses provided evidence of cartilage and osteoid tissue inside the diffusion chambers, whereas bone was also observed inside the DBM implants. In conclusion, this experimental procedure is capable of selecting a cell population from bone marrow which, in the presence of rhOP-1, achieves skeletogenic potential under in vitro as well as in vivo environments.

Promotion of calvarial cell osteogenesis by endothelial cells.

Bone development and remodeling are associated with changes in the pattern of vascularization. Here we show that endothelial cells isolated from rat liver or bovine aorta can greatly enhance bone formation when implanted in diffusion chambers with rat fetal calvarial cells. The latter cells are unable to form bone when implanted alone at low initial cell density. The amount of mineralization measured by calcium deposition was 70 times higher in chambers containing calvarial cells mixed with endothelial cells from isologous liver or bovine aorta than in chambers containing endothelial or calvarial cells alone. Alkaline phosphatase activity was increased 20-fold. Calvarial cells in the presence of demineralized bone matrix powder did not form bone when implanted under similar conditions. Endothelial cells implanted alone seemed to enhance neovascularization around the Millipore diffusion chambers.

Retinoic acid and glucocorticoids enhance the effect of 1,25-dihydroxyvitamin D3 on bone gamma-carboxyglutamic acid protein synthesis by rat osteosarcoma cells.

Two 1,25-dihydroxyvitamin D3-controlled parameters in the osteoblastlike osteosarcoma cell line ROS 17/2, bone gamma-carboxyglutamic acid-containing protein (BGP) and collagen synthesis, were measured after pretreatments with either retinoic acid (RA), or triamcinolone acetate (TRM). RA and TRM both caused double the expected increase in BGP secretion at 16 hr after treatment with 1,25-dihydroxyvitamin D3. Triamcinolone acetate concentrations of 10(-8) and 10(-9) M or 10(-6) M retinoic acid were effective in enhancing the 1,25-dihydroxyvitamin D3 stimulation of BGP secretion. Treatment with RA or TRM alone did not stimulate BGP secretion. RA alone had no effect on BGP secretion, while TRM inhibited BGP secretion. Collagen synthesis is inhibited by 1,25-dihydroxyvitamin D3. Neither retinoic acid nor triamcinolone acetate enhanced the 1,25-dihydroxyvitamin D3-mediated inhibition of collagen synthesis. Retinoic acid by itself inhibited collagen synthesis but did not change the 1,25 dihydroxyvitamin D3-mediated inhibition of collagen synthesis. Triamcinolone acetate by itself or together with 1,25-dihydroxyvitamin D3 increased collagen synthesis. We conclude that, although both triamcinolone acetate and retinoic acid increase the 1,25-dihydroxyvitamin D3 stimulation of BGP secretion by ROS 17/2 cells, they have different effects on the regulation of collagen production. Thus, although both hormones increase the 1,25-dihydroxyvitamin D3 receptor concentration in these cells, their actions are not mediated solely by this mechanism.

Demineralized bone matrix mediates differentiation of bone marrow stromal cells in vitro: effect of age of cell donor.

Bone maintenance requires a continuous source of osteoblasts throughout life. Its remodeling and regeneration during fracture repair is ensured by osteoprogenitor stem cells which are part of the stroma of the bone marrow (BM). Many investigators have reported that in cultured BM stromal cells there is a cell population that will differentiate along an osteogenic lineage if stimulated by the addition of osteogenic inducers, such as dexamethasone (dex), beta-glycerophosphate (beta-GP), transforming growth factor beta-1 (TGF-beta 1) and bone morphogenetic protein-2 (BMP-2). Here we report the effects of demineralized bone matrix (DBM) on the osteogenic differentiation of BM stromal cells in vitro, using morphological criteria, alkaline phosphatase (AP) activity, and calcium accumulation. DBM and DBM-conditioned medium (DBMcm) enhanced bone formation in the presence of dex and beta-GP, whereas DBM particles caused changes in the cell phenotype. Temporal expression of total and skeletal AP by BM stromal cells from 4-week-old rats showed a biphasic pattern enhanced by DBM and suggesting the presence of two cell populations. In one population, AP synthesis reaches a maximum during the first week in culture, following which cells either die or loose their ability to synthesize AP. A second, less abundant population begins to proliferate and synthesize AP during the second and third weeks. The synthesis of AP, which often decreases by the third week, can be maintained at high levels only if DBM is added to the cultures. BM stromal cells isolated from 24- and 48-week-old rats showed a decrease or loss of this biphasic AP expression pattern compared with cells isolated from 4-week-old rats. The addition of DBM to cultures derived from 24- and 48-week-old rats stimulated mostly the second cell population to synthesize AP, suggesting that DBM contains a factor(s) that acts on a specific bone marrow cell population by increasing the proliferation of active cells or inducing the differentiation of dormant cells.

Capture and expansion of bone marrow-derived mesenchymal progenitor cells with a transforming growth factor-beta1-von Willebrand's factor fusion protein for retrovirus-mediated delivery of coagulation factor IX.

Mesenchymal stem cells give rise to the progenitors of many differentiated phenotypes, including osteocytes, chrondocytes, myocytes, adipocytes, fibroblasts, and marrow stromal cells, which are capable of self-renewal and undergo expansion in the presence of transforming growth factor-beta1 (TGF-beta1). The present study was designed to test the concept that mesenchymal progenitor cells could be selected and expanded by virtue of their intrinsic physiologic responses to TGF-beta1. Human bone marrow aspirates were initially cultured, under low serum conditions, in collagen pads or gels impregnated with a genetically engineered TGF-beta1 fusion protein bearing an auxiliary von Willebrand's factor-derived collagen-binding domain (TGF-beta1-vWF). Histologic examination of TGF-beta1-vWF-supplemented collagen pads from 8-day cultures revealed the selective survival of a population of mononuclear blastoid cells. The TGF-beta-responsive cells were expanded to form stromal/fibroblastic colonies by serum reconstitution, and further to form osteogenic colonies upon supplementation with osteoinductive factors. In comparative studies, both marrow-derived progenitor cells and mature stromal cells were transduced with a retroviral vector bearing a human factor IX construct. Both the transduced progenitor cells and mature stromal cells expressed the factor IX transgene at levels comparable to those reported for human fibroblasts. Transplantation of murine progenitor cells bearing the human factor IX vector into syngeneic B6CBA mice resulted in detectable circulating levels of the human factor IX antigen. Taken together, these data demonstrate a novel physiologic approach for the selection of mesenchymal precursor cells followed by mitotic expansion, transduction, and transplantation of these progenitor cells with retroviral vectors bearing therapeutic genes.

Stimulation of fibroblast biosynthetic activity by serum of patients with pretibial myxedema.

Skin fibroblasts from the shoulder and lower extremities of normal individuals, as well as from patients with pretibial myxedema (PTM) were grown in culture. When cells reached the monolayer stage, they were labeled with 3H-glucosamine and tested for hyaluronic acid synthesis in the presence of either serum from PTM patients or normal human serum. All the fibroblasts from the pretibial area synthesized 2 to 3 times more hyaluronic acid when incubated with PTM sera than when incubated in normal human serum. Fibroblasts cultured from skin of the back or prepuce did not respond to PTM sera. This heat-stable, protease-sensitive, and dialyzable, fibroblast-stimulating factor is not a 7S gamma-globulin. The enhanced sensitivity to PTM sera exhibited by fibroblasts from the lower extremities may explain why the lesions in this disease are restricted primarily to that area.

Elevated levels of plasminogen activator inhibitor-1 may account for the altered fibrinolysis by keloid fibroblasts.

Using a 3-dimensional fibrin gel model system simulating fibroplasia of wound repair, we investigated the interaction between keloid fibroblasts and fibrin matrix and compared it with that of normal fibroblasts. Normal skin fibroblasts caused fibrin gel degradation under serum-free conditions, whereas keloid fibroblasts did not cause microscopically detectable gel degradation. Fibrin gel degradation occurred through plasmin-mediated fibrinolysis, which was initiated by fibroblasts exhibited high uPA but low plasminogen activator inhibitor-1 (PAI-1) activities, and transforming growth factor-beta 1 prevented fibrinolysis of normal fibroblasts by upregulating PAI-1 while downregulating uPA activities. In contrast, keloid fibroblasts exhibited an intrinsically high level of PAI-1 and a low level of uPA. This change in the ratio of activator and inhibitor activities was attributed to altered fibrin degradation by keloid fibroblasts. The PAI-1 increase was also demonstrated at the RNA level by Northern analysis. In terms of the pivotal role of the plasmin/plasminogen activator system in matrix remodeling, the elevated PAI-1 level exhibited by keloid fibroblasts may have significant consequences not only in altered fibrin degradation, but also in subsequent repair steps that lead to keloids and fibrosis.

Crosslinked CNBr-activated hyaluronan-collagen matrices: effects on fibroblast contraction.

Previously we demonstrated that the contraction of collagen fibrillar matrices (CFM) by fibroblasts was significantly reduced when high concentrations (> 1 mg/ml) of hyaluronan were present in the media. This highlights the prominent role of hyaluronan in preventing scar formation during fetal wound healing. In the present study, we covalently crosslinked different concentrations of CNBr-activated hyaluronan to CFM and investigated the ability of fibroblasts to contract these matrices. Matrices are not contracted when optimal concentrations of CNBr-activated hyaluronan are crosslinked to collagen. Biochemical and cytological analysis of these matrices suggests that hyaluronan strengthens the collagen fibrils and blocks direct communication between fibroblasts and the collagen fibrils. Based on current evidence we are able to propose two molecular crosslinking models, multiple point attachment and simple point attachment between CNBr-activated hyaluronan and collagen molecules, hypotheses supported by the susceptibility of these matrices to digestion by collagenase.

A new model for heterotopic aortic valve transplantation.

A new model for heterotopic aortic valve transplantation in the rat is described. A composite allograft with an intact aortic valve and partial mitral valve was harvested from 4-month-old (400-450 g) Long-Evans rats and grafted heterotopically into the abdominal aorta of 4-week-old (80-100 g) rats with an optimal size match. At the end of a 1-month observation period, all experimental animals were alive and all showed 100% patency of the aortic valve allografts on microscopic evaluation after death (n=40). Unlike previously used methods, the proposed model allows for the preservation of all three aortic valve cusps and a more remote placement of the anastomotic suture line from the aortic valve annulus. The use of younger recipient rats improves size match and amplifies allograft calcification. The purpose of this study was to provide an animal model to evaluate modalities of preservation and chemical treatment for aortic valves used as allografts or bioprosthesis.

Stability of collagen phenotype in morphologically modulated rabbit corneal endothelial cells.

Primary monolayer cultures of rabbit corneal endothelial cells maintained characteristic polygonal morphology and synthesized type IV collagen as a major collagenous peptide. Upon serial passage, [3H]-proline incorporation into proteins gradually decreased from the secondary subculture to a low of 71% of that in the primary culture in the fourth subculture, while type IV collagen synthesis significantly decreased from the tertiary subculture, and the fourth subculture retained only 22% of the primary culture value. When collagen molecules synthesized by the fourth subculture progeny were analyzed by SDS electrophoresis, no alteration was noted. The fourth subculture progeny continued to synthesize the characteristic type IV collagen, which migrates slightly faster than the beta 12 (I) band on SDS electrophoresis. However, the cellular morphology was changed dramatically from the characteristic polygonal shape to enlarged cells in the fourth subculture and mitotic activity was no longer apparent. Immunofluorescence studies showed that the enlarged fourth subculture progeny stained with anti-IV collagen antibodies, as did the primary polygonal endothelial cells. These findings confirmed type IV collagen synthesis in the enlarged endothelial cells. In addition, the morphologically altered cells continuously deposited Descemet's membrane-like extracellular matrices between the basal cell layer and the plastic substratum. These results suggest that endothelial cells lose their proliferative capacity as they age, but compensate for the loss of cell numbers by enlargement while maintaining a normal collagen phenotype.

Modulation of endothelial cell morphology and collagen synthesis by polymorphonuclear leukocytes.

Primary corneal endothelial monolayers exposed to polymorphonuclear leukocytes undergo a series of morphologic alterations. Elongation occurred in foci within 3 days after removal of polymorphonuclear leukocytes, the modulated endothelial foci grew into fibroblastic colonies, and the fibroblastic cells eventually overgrew the endothelial cells. Control cultures of endothelial cells originated from confluent monolayers became enlarged, attenuated and lost their characteristic polygonal shape within 10 days following postconfluency , but no fibroblastic changes were seen. "Wounding" the endothelial monolayer with a focal freeze resulted in death of cells with slow regeneration. In the presence of polymorphonuclear leukocytes, cell migration into the wound was enhanced, and there was selective proliferation of fibroblastic cells. Indirect immunofluorescent studies showed that anti-type I collagen antibodies stained the fibroblastic foci in the polymorphonuclear leukocyte-treated endothelial cells and the fully modulated endothelial cells. The fully modulated cells also showed loss of contact inhibition leading to mutilayering of cells and extracellular matrices, which accumulated not only between the basal cell layers and plastic substratum but also in the cellular interstices. When collagen phenotype was analyzed by SDS electrophoresis in comparison with corneal endothelial phenotypes (type IV collagen), type I procollagen synthesis became evident in the secondary subculture originated from polymorphonuclear leukocyte-treated endothelial cells. Limited pepsin treatment gave rise to type I collagen as a major collagenous peptide. Polymorphonuclear leukocytes, thus, apparently contribute to the modulation of endothelial cells into fibroblastic cells, which also switch their collagen phenotype from type IV to type I collagen synthesis.

A new technique for isolation of Descemet's membrane: preliminary studies.

The structure and function of basement membranes have been the subject of extensive investigation. The present study takes advantages of a new experimental procedure to yield ultrastructurally pure basement membranes and applies this methodology to Descemet's membrane, a highly specialized ocular basement membrane. Rabbit Descemet's membranes and associated endothelial cells were mechanically isolated without contaminating stromal elements. The endothelial cells were then solubilized and removed by treatment with detergents as verified by light microscopy and scanning and transmission electron microscopy. The Descemet's membrane remained intact and retained its fibrillar fine structure. Therefore purity of starting material for ongoing morphological and biochemical studies of isolated Descemet's membranes is demonstrated. These investigations will provide a valuable data base for comparison with disease-altered Descemet's membranes.

Type I collagen and fibronectin synthesis by retrocorneal fibrous membrane.

The primary cultures obtained from the experimentally induced retrocorneal fibrous membrane synthesized and secreted into the medium mainly type I procollagen. This collagen was characterized after limited pepsin treatment and identified as type I collagen by the following criteria: (1) it contained two alpha 1 chains and one alpha 2 chain, (2) its sedimentation behavior was identical to that of type I collagen from skin, and (3) its peptide map after limited proteolysis with Staphylococcus aureus V8 protease was identical to that of type I collagen. The medium contained procollagen I, which was converted into alpha size chains by limited pepsin treatment, whereas the cellular fraction contained type I collagen already processed to its end product. Type III collagen and basement membrane collagen were present as minor components in this system Fibronectin, one of the major glycoproteins in extracellular matrices, was also synthesized and secreted into the medium. In contrast, normal corneal endothelial cells produce mainly basement membrane collagen.

Polypeptide growth factors: targeted delivery systems.

Growth factors are becoming extremely valuable tools in our attempts to understand the mechanisms that modulate cellular activities. Their targeting to appropriate cells and maintaining adequate pharmacological levels becomes essential, particularly in view of the different effects that these compounds have on various cells and the dose dependence of their response. Within this context, this review focuses primarily on the delivery of growth factors involved in the processes of wound healing and tissue repair.

Modulation of osteogenesis by isolated calvaria cells: a model for tissue interactions.

Dispersed cells from calvaria fetuses when suspended in agarose deposit a metachromic matrix. Anchorage independence is a requirement for these cells to express type II collagen. Type I collagen is preferentially expressed by cells in monolayer cultures. Cell separation by isopycnic percoll gradient showed that cells recovered from densities 1.04 g ml-1 or higher synthesized type II collagen when suspended in agarose. These cells cultured on a plastic surface expressed type I collagen. Endothelial cells isolated from rat liver or bovine aorta when implanted in diffusion chambers together with dispersed calvaria cells enhanced the formation of bone. The calcium content was 70 times higher than in chambers containing either endothelial or calvaria cells alone. The former cells developed no bone at all when implanted alone, even in the presence of demineralized bone matrix, but some isolated islands of cartilage could be seen.

Cellular events associated with the induction of bone by demineralized bone.

Implantation of demineralized bone (DB) in the form of powder or intact segments in extra skeletal sites stimulates new bone formation. Urist and co-workers presented substantial evidence that there is a noncollagenous protein that has the ability to induce bone formation. One aim of this study was to trace the process of bone formation when DB, in the form of perforated rectangular plates, is implanted subcutaneously in 2-month-old rats. A second objective was to determine whether cartilage cells play a role in the formation of bone in this model. Various DB plates with 0.25 mm diameter holes were implanted subcutaneously for 1-4 weeks in rats. One week after implantation, DB plates were covered by vascularized connective tissue that invaded the perforations. Aggregates of chondrocytes were observed within the holes and on periosteal surfaces in only a few specimens. Further cartilage proliferation was not observed, and by the 2nd week there was no evidence of endochondral bone formation. Where these cartilage-like cells were present, a thin layer of mineral was deposited around them; resorption and fibrous tissue infiltration followed. This aborted form of endochondral calcification was not followed spatially by bone formation. Patent vascularized channels were invaded by alkaline phosphatase-positive mononuclear cells and fibroblasts, and became enlarged by the enzymatic action of macrophages. The next step involved the calcification of DB plates adjacent to the wide spaces. Osteoclasts now appeared leading to the resorption of this recalcified matrix. The eroded and now enlarged lacunar surfaces were lined by newly formed bone and osteoblasts. This process continued so that, at the end of 4 weeks following implantation, the original DB plates were replaced by trabecular bone. Biochemical data on calcium and alkaline phosphatase levels in the implants paralleled the morphological observations.

Effects of indomethacin on demineralized bone-induced heterotopic ossification in the rat.

Indomethacin inhibits bone formation when treatment is initiated before the implantation of demineralized bone matrix (DBM). For the inhibition of bone induction to occur, indomethacin treatment had to be initiated 6 h or more before implantation of DBM. Initiating the drug treatment at or after the time of DBM implantation had no effects on the amounts of new bone formed. The inhibition by indomethacin is dose related over a range between 0.04 and 4 mg/kg body weight. Recovered day-1 DBM implants, transplanted into indomethacin pre- and posttreated syngeneic rats, formed bone at the same rate as controls did. However, recovered day-1 DBM implants lyophilized before transplantation showed decreased bone formation but significant dystrophic calcification as judged by a lower alkaline phosphatase activity and an elevated calcium content.