Tendon tissue engineering: adipose-derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems.

Tendon tissue engineering with a biomaterial scaffold that mimics the tendon extracellular matrix (ECM) and is biomechanically suitable, and when combined with readily available autologous cells, may provide successful regeneration of defects in tendon. Current repair strategies using suitable autografts and freeze-dried allografts lead to a slow repair process that is sub-optimal and fails to restore function, particularly in difficult clinical situations such as zone II flexor tendon injuries of the hand. We have investigated the effect of GDF-5 on cell proliferation and gene expression by primary rat adipose-derived stem cells (ADSCs) that were cultured on a poly(DL-lactide-co-glycolide) PLAGA fiber scaffold and compared to a PLAGA 2D film scaffold. The electrospun scaffold mimics the collagen fiber bundles present in native tendon tissue, and supports the adhesion and proliferation of multipotent ADSCs. Gene expression of scleraxis, the neotendon marker, was upregulated seven- to eightfold at 1 week with GDF-5 treatment when cultured on a 3D electrospun scaffold, and was significantly higher at 2 weeks compared to 2D films with or without GDF-5 treatment. Expression of the genes that encode the major tendon ECM protein, collagen type I, was increased by fourfold starting at 1 week on treatment with 100 ng mL(-1) GDF-5, and at all time points the expression was significantly higher compared to 2D films irrespective of GDF-5 treatment. Thus stimulation with GDF-5 can modulate primary ADSCs on a PLAGA fiber scaffold to produce a soft, collagenous musculoskeletal tissue that fulfills the need for tendon regeneration.

Treatment with insulin-like growth factor-1 increases chondrogenesis by periosteum in vitro.

The repair of defects in articular cartilage with hyaline tissue that is resilient to wear is a challenging problem. Fibrocartilaginous tissue forms in response to injury through the articular surface and degenerates under mechanical load. Because periosteum contains cells, which are capable of synthesizing cartilage matrix proteins, it has been used to repair defects in articular surfaces. Treatment of periosteal grafts with growth factors, particularly those that elicit chondrocyte gene expression, may improve tissue regeneration. Gene expression by periosteal explants in vitro was measured. Expression of type II collagen and aggrecan mRNA was increased in response to treatment with IGF-I. Furthermore, IGF-I treatment caused an increase in type II collagen and aggrecan mRNA that was time and concentration dependent. The effect of short and long-term (continuous) incubations was compared to determine if a pretreatment could be used to condition a graft for subsequent surgical use. Short-term incubation in vitro with IGF-I followed by incubation without IGF-I was nearly as effective at increasing expression of type II collagen and aggrecan mRNA as incubation for the same length of time with IGF-I present continuously in the culture media. Treatment with IGF-I also produced cell clustering and nodule formation which are indicative of chondrogenesis. These results suggest that pretreatment with IGF-I in vitro may enhance the effectiveness of a graft to produce hyaline cartilage in vivo. Whether the cellular and molecular changes we have observed can lead to the formation of tissue that withstands the mechanical forces exerted by weight bearing remains to be determined.

Comparison of lumbar spine fusion using mixed and cloned marrow cells.

STUDY DESIGN: Prospective study on lumbar spine fusion using cloned and mixed marrow cells. OBJECTIVE: To analyze the effectiveness of cloned osteoprogenitor cells in spine fusion and their differentiation in vivo using a traceable gene. SUMMARY OF BACKGROUND DATA: Although autografts are currently the standard for stable spine fusion, supply is limited. Alternative graft materials need to be developed and evaluated. METHODS: An osteoprogenitor cell, D1-BAG, cloned from mouse bone marrow and transduced with LacZ and neomycin resistance genes, and mixed marrow stromal cells from marrow blowouts were used in athymic rats to establish posterior spinal fusion; 2 x 10(6) cells in 100 microL Matrigel were implanted into the lumbar fusion bed in 36 animals, whereas Matrigel without cells was used in 16 animals as control. Rats were killed at 2, 3, 6, and 9 weeks, and the spines were evaluated by manual palpation, radiographs, and histology. RESULTS: Two weeks after surgery radiopaque tissue was seen at transplantation sites with D1-BAG cells but not at sites with mixed marrow stromal cells. Successful spine fusion at 6 and 9 weeks was observed in 8 of 8 (100%) animals receiving DI-BAG cells, 4 of 8 (50%) in mixed marrow stromal cells, and 0 of 8 (0%) in control animals. CONCLUSIONS: Compared with mixed marrow stromal cells, cloned osteoprogenitor cells can produce a larger amount of mature osseous tissue at an earlier time point during spine fusion.

Pluripotential mesenchymal cells repopulate bone marrow and retain osteogenic properties.

Precursor cells, isolated from bone marrow, can develop into various cell types and may contribute to skeletal growth, remodeling, and repair. The D1 cell line was cloned from a multipotent mouse bone marrow stromal precursor and has osteogenic, chondrogenic, and adipogenic properties. The osteogenic phenotype of these precursor cells is relevant to the process of fracture healing and osteointegration of prosthetic implants. The D1 cells were labeled genetically using a replication incompetent retroviral vector encoding beta-galactosidase, an enzyme which is used as a marker. Labeled cells are readily identifiable by staining with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside and by flow cytometry, and retain the desired osteogenic characteristics in vivo as shown by von Kossa staining, alkaline phosphatase assay, an increase in cyclic adenosine monophosphate in response to parathyroid hormone, osteocalcin messenger ribonucleic acid production, and bone formation in diffusion chambers. In addition, the cells cloned from marrow stroma repopulate the marrow of host mice, persist for several weeks, and retain their osteogenic potential ex vivo. The data suggest that such cells may be used to replenish the number of osteoprogenitors in marrow, which appear to decrease with age, thereby leading to recovery from bone loss and improved bone growth and repair. Labeling these cells creates a model in which to study the potential of such cells to participate in fracture repair, ingrowth around prosthetic implants, treatment of osteoporosis, and to explore the possibility of gene delivery to correct mutations or defects in metabolism that are responsible for certain skeletal abnormalities.

Alterations of cartilage and collagen expression during fracture healing in experimental diabetes.

We investigated alterations in the expression of mRNA for type II and type X collagen in fracture callus of experimentally induced diabetic animals compared with controls and performed radiographic, histological, immunocytochemical and biomechanical studies. Experimentally induced diabetic rats exhibited an alteration in the temporal expression of type II and type X collagen mRNA and a decrease in type X mRNA expression as compared to controls. Radiographs showed a more intense periosteal reaction and a more rapid reconstitution of cortices in control versus diabetic animals. Histologically there was a delay in chondrocyte maturation and hypertrophy seen in diabetics. Immunolocalization of type X collagen demonstrated a delay in type X collagen expression around the hypertrophic chondrocytes. Biomechanical analysis showed a decrease in the strength of healing fractures in diabetic animals. Fracture healing in diabetic patients is compromised and may lead to delays in bone union. Though the exact mechanisms are unknown, we present evidence of decreased mechanical strength of the fracture and suggest that associated changes in collagen expression and chondrocyte maturation are mechanisms leading to delayed healing in untreated and poorly controlled diabetes.

Marrow stromal cells embedded in alginate for repair of osteochondral defects.

Articular cartilage defects of sufficient size ultimately degenerate with time, leading to arthritic changes. Numerous strategies have been used to address full-thickness cartilage defects, yet none thus far has been successful in restoring the articular surface to its preinjury state. We compared the effects of agarose, alginate, and type I collagen gels on the expression of cartilage-specific markers from rabbit marrow stromal cells and then assessed the in vivo effects of cells seeded in alginate beads on the repair of full-thickness osteochondral defects in the rabbit model. Marrow aspirates from rabbits were cultured and the stromal population selected. Marrow stromal cells were then placed in either 1.2% w/v alginate, type I collagen gels (3 mg/mL), or 0.5% agarose suspension culture. After 2, 5, 10, and 20 days in culture, the RNA was extracted and analyzed by reverse transcription polymerase chain reaction for the cartilage-specific markers aggrecan and type II collagen. The strongest increase in aggrecan and type II collagen gene expression was found in the agarose suspension followed by alginate; type I collagen gels induced the lowest levels. Alginate beads were chondrogenic and maintained their size and consistency over time in culture, whereas the cell-seeded collagen gels invariably contracted. Full-thickness defects measuring 3 x 6 mm x 3 mm deep were then created in the medial femoral condyles of rabbit knees and filled with alginate beads, alginate beads seeded with stromal cells, or left empty. Alginate beads containing stromal cells remained within the defects and progressively filled the defects with regenerate tissue. Histologic analysis showed viable, phenotypically chondrogenic cells in the defects. The matrix stained positive with safranin O, indicating proteoglycan synthesis, and bonding between the regenerate and host tissue was excellent. We have shown quantitative differences in the chondrogenic effects of the biomaterials tested. Alginate induces the chondrogenic phenotype in marrow stromal cells in vitro, and possesses the necessary physical characteristics and handling properties to support cells and serve as a carrier to fill full-thickness osteochondral defects in vivo.

Pluripotential marrow cells produce adipocytes when transplanted into steroid-treated mice.

The effect of steroids on adipogenesis by D1-BAG, a pluripotent cell cloned from mouse bone marrow and transfected with traceable genes encoding beta-galactosidase and neomycin resistance, was investigated in vitro in culture and in vivo after injection into mice. Treatment of D1-BAG cells in culture with dexamethasone produced an accumulation of lipid vesicles and stimulated expression of the fat cell-specific 422(aP2) mRNA. Fifty-six mice each received 1 x 10(6) D1-BAG cells, either by tail-vein injection or by direct injection into the marrow of the right femur. Another 38 mice received either saline injection or no treatment as controls. Half of the animals in each group were treated with 3 mg/kg of methylprednisolone per week. Analysis of marrow blow-outs by flow cytometry, DNA analysis by PCR, and X-gal stain of histological sections indicated that cells transplanted by either intravenous or intramedullary injection had appeared and persisted in the marrow of host mice. Cell sorting by flow cytometry and staining with Sudan IV demonstrated that steroid treatment produced adipogenesis in 5-9% of transplanted cells. The results indicate that steroid-induced differentiation of potentially osteogenic marrow cells into adipocytes in vivo may contribute to the development of osteoporosis and osteonecrosis.

The Nicolas Andry award. The pathogenesis and prevention of steroid-induced osteonecrosis.

The effects of steroids on a cloned pluripotential cell from bone marrow stroma were examined in vitro in culture and in vivo after the cells were transfected with a traceable gene and transplanted into host mice. Bipedal chickens were treated with steroids to establish a model for osteonecrosis. The effects of a lipid lowering agent, lovastatin, on the prevention of steroid induced adipogenesis in vitro in cell culture, and on adipogenesis and osteonecrosis in vivo in chickens, were evaluated. On treatment with dexamethasone, cloned pluripotential cells began to differentiate into adipocytes and expressed a fat specific gene, whereas the expression of Type I collagen and osteocalcin messenger ribonucleic acid decreased. Addition of lovastatin in culture inhibited steroid induced fat gene expression and counteracted the inhibitory effect of steroids on osteoblastic gene expression. Cloned pluripotential cells were transduced with a traceable retrovirus vector encoding the beta-galactosidase and neomycin resistance genes. The transfected cells were administered to mice either by tail vein or by direct intramedullary injection. Half of the animals in each group were treated with steroids. Histologic sections showed the appearance of transplanted cells in the marrow. Analysis of marrow blowouts by flow cytometry revealed that steroid treatment produced adipogenesis in transplanted cells. Evidence of osteonecrosis was observed in steroid treated chickens, whereas sections from animals treated with steroids and lovastatin showed less adipogenesis and no bone death. The results indicate that steroid induced adipogenesis in the marrow may contribute to osteonecrosis and that lovastatin may be helpful in preventing the development of steroid induced osteonecrosis.

Comparison of surgically attached and non-attached repair of the rat Achilles tendon-bone interface. Cellular organization and type X collagen expression.

The effects of surgical repair versus non-repair on cell morphology and type X collagen expression were investigated using a rat model of Achilles tendon avulsion. The animals were divided into four groups. In Group 1, tendon was reattached to the original attachment site by suturing through a drill hole in the calcaneus; in Group II, tendon was not reattached and a drill hole was not made; in Group III, tendon was not reattached but a drill hole was made; and the animals in Group IV were sham operated. In Group I (tendon reattached), at 2 weeks postoperatively, many hypertrophic chondrocytes appeared at the reattachment site adjacent to bone and type X collagen was detected immunologically both in the cells and in the extracellular matrix. After 4 weeks, the cells at the original site of attachment were arranged in rows along the newly formed tendon fibers and were stained with type X collagen antibody. By contrast, when tendon was not reattached (Groups II and III), a gap between the original attachment site and the tendon stump was observed through the entire postoperative period. At 8 weeks, the original attachment site was covered by fibrocartilaginous tissue and tendon became attached to the calcaneal fibrocartilage area, which is proximal to the original attachment site. Type X collagen was detected in the cells which were adjacent to bone. In Group IV (sham operation), there were no changes in histology or type X collagen distribution, either at the attachment site or in tendon and bone, compared with the non-operated control rats. These results suggest that surgical reattachment of tendon to the original site is important to help reorganize cells during the repair process. Type X collagen was identified immunohistochemically in the cells adjacent to bone in all the groups, suggesting that it may play a role in maintaining distinct areas of calcified and non-calcified fibrocartilage.

Role of the pro-alpha2(I) COOH-terminal region in assembly of type I collagen: truncation of the last 10 amino acid residues of pro-alpha2(I) chain prevents assembly of type I collagen heterotrimer.

Procollagen (Type I) contains a noncollagenous COOH-terminal propeptide (C-propeptide) hypothesized to be important in directing chain association and alignment during assembly. We previously expressed human pro-alpha2(I) cDNA in rat liver epithelial cells, W8, that produce only pro-alpha1(I) trimer collagen (Lim et al. [1994] Matrix Biol. 14: 21-30). In the resulting cell lines, alpha2(I) assembled with alpha1(I) forming heterotrimers. Using this cell system, we investigated the importance of the COOH-terminal propeptide sequence of the pro-alpha2(I) chain for normal assembly of type I collagen. Full-length human pro-alpha2(I) cDNA was cloned into expression vectors with a premature stop signal eliminating the final 10 amino acids. No triple-helical molecules containing alpha2(I) were detected in transfected W8 cells, although pro-alpha2(I) mRNA was detected. Additional protein analysis demonstrated that these cells synthesize small amounts of truncated pro-alpha2(I) chains detected by immunoprecipitation with a pro-alpha2(I) antibody. In addition, since the human-rat collagen was less thermostable than normal intraspecies collagen, wild-type and C-terminal truncated mouse cDNAs were expressed in mouse D2 cells, which produced only type I trimers. Results from both systems were consistent, suggesting that the last 10 amino acid residues of the pro-alpha2(I) chain are important for formation of stable type I collagen.

Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reaction by prostate-specific antigen-producing tumors in athymic and SCID/bg mice using LNCaP and lineage-derived metastatic sublines.

LNCaP lineage-derived human prostate cancer cell lines C4-2 and C4-2B4 acquire androgen independence and osseous metastatic potential in vivo. Using C4-2 and C4-2B4 the goals of the current investigation were 1) to establish an ideal bone xenograft model for prostate cancer cells in intact athymic or SCID/bg mice using an intraosseous route of tumor cell administration and 2) to compare prostate cancer metastasis by administering cells either through intravenous (i.v.) or intracardiac administration in athymic or SCID/bg mice. Subsequent to tumor cell administration, prostate cancer growth in the skeleton was assessed by radiographic bone density, serum prostate-specific antigen (PSA) levels, presence of hematogenous prostate cancer cells and histopathologic evaluation of tumor specimens in the lymph node and skeleton. Our results show that whereas LNCaP cells injected intracardially failed to develop metastasis, C4-2 cells injected similarly had the highest metastatic capability in SCID/bg mice. Retroperitoneal and mediastinal lymph node metastases were noted in 3/7 animals, whereas 2/7 animals developed osteoblastic spine metastases. Intracardiac injection of C4-2 in athymic hosts produced spinal metastases in 1/5 animals at 8-12 weeks post-injection; PC-3 injected intracardially also metastasized to the bone but yielded osteolytic responses. Intravenous injection of either LNCaP or C4-2 failed to establish tumor colonies. Intrailiac injection of C4-2 but not LNCaP nor C4-2B4 cells in athymic mice established rapidly growing tumors in 4/8 animals at 2-7 weeks after inoculation. Intrafemoral injection of C4-2 (9/16) and C4-2B4 (5/18) but not LNCaP (0/13) cells resulted in the development of osteoblastic bone lesions in athymic mice (mean: 6 weeks, range: 3-12 weeks). In SCID/bg mice, intrafemoral injection of LNCaP (6/8), C4-2 (8/8) and C4-2B4 (8/8) cells formed PSA-producing, osteoblastic tumors in the bone marrow space within 3-5 weeks after tumor cell inoculation. A stepwise increase of serum PSA was detected in all animals. Reverse transcription-polymerase chain reaction (RT-PCR) to detect hematogenously disseminated prostate cancer cells could not be correlated to either serum PSA level or histological evidence of tumor cells in the marrow space. We have thus established a PSA-producing and osteoblastic human prostate cancer xenograft model in mice.

The Otto Aufranc Award. Lovastatin prevents steroid induced adipogenesis and osteonecrosis.

Osteonecrosis of the femoral head was induced experimentally in chickens after the administration of a high dose of corticosteroids. Lovastatin was used to prevent the effects of the steroid on adipogenesis in cultured cells, and adipogenesis and osteonecrosis in chickens. The in vitro study, with marrow cells in culture, showed that Lovastatin inhibited steroid induced fat specific gene expression and counteracted the inhibitory effects of steroids on osteoblastic gene expression. For the in vivo study, 83 adult chickens were used: 48 received methylprednisolone 3 mg/kg weekly via intramuscular injection (Group A). Fifteen received the steroid (as in Group A) plus Lovastatin 20 mg per animal per day orally (Group B). Ten chickens received Lovastatin only (Group C). Another 10 received no medication and served as the control group (Group D). Evidence of osteonecrosis was observed in specimens from Group A, including subchondral bone death and resorption, fat cell proliferation, and new bone formation. Conversely, sections from Group B showed less adipogenesis and no bone death. It is concluded that the bipedal chicken is a useful animal model for studies of osteonecrosis and that lipid clearing agents, such as Lovastatin, may be helpful in preventing the development of steroid induced osteonecrosis.

Steroid-induced adipogenesis in a pluripotential cell line from bone marrow.

UNLABELLED: We studied the effect of steroids on the differentiation of a pluripotential mesenchymal cell with use of a cell line (D1) from mouse bone-marrow stroma. The cells were treated with increasing (10[-9], 10(-8), and 10(-7)-molar) concentrations of dexamethasone for increasing durations ranging from forty-eight hours to twenty-one days. The appearance of triglyceride vesicles in the cells indicated that this treatment had induced the differentiation of the cell into adipocytes. The number of cells that contained the triglyceride vesicles and the expression of a fat-cell-specific gene, 422(aP2), increased with longer durations of exposure to dexamethasone and with higher concentrations of the steroid. Treatment with dexamethasone also diminished the expression of alpha1 type-I collagen mRNA and osteocalcin mRNA. The data indicate that dexamethasone stimulates the differentiation of cells in bone-marrow stroma into adipocytes as well as the accumulation of fat in the marrow at the expense of expression of type-I collagen and osteocalcin mRNA, thereby suppressing differentiation into osteoblasts. CLINICAL RELEVANCE: Steroid-induced adipogenesis by bone progenitor cells in marrow may influence the development of osteonecrosis. It is therefore important to consider the investigation of a treatment, such as the inhibition of the metabolism and accumulation of fat in marrow, that can prevent the onset of osteonecrosis.

Changes in the expression of type-X collagen in the fibrocartilage of rat Achilles tendon attachment during development.

This study histologically and immunohistochemically demonstrated developmental changes in cell morphology and expression of type-X collagen in the attachment of the Achilles tendon to the calcaneus in the rat. Although the site of attachment in the mature rat showed a well organized, direct insertion that was composed of tendon, fibrocartilage, calcified fibrocartilage, and bone, this four-zone structure was not observed in the immature 1-week-old rat. Formation of fibrocartilage was observed at 2 weeks, together with the hypertrophy of chondrocytes and the appearance of the secondary center of ossification. Type-X collagen was not detected either in chondrocytes in the attachment area at 1 week or in hypertrophic chondrocytes at the attachment at 2 weeks. In the 3-week-old rat, the secondary center of ossification extended to the area of attachment and type-X collagen was detected both in cartilage spicules within the secondary center of ossification and in cells found at the attachment adjacent to the secondary center of ossification. A four-zone structure had been established by 6 weeks and remained through 20 weeks. After 6 weeks, type-X collagen was identified both in the attachment of the tendon and beneath the calcaneal fibrocartilage. Type-X collagen is produced by cells in transitional zones between calcified and noncalcified tissue, such as the interface between articular cartilage and subchondral bone. In these areas, the expression of this protein persists through maturity and is not transient.

Dexamethasone promotes von kossa-positive nodule formation and increased alkaline phosphatase activity in costochondral chondrocyte cultures.

This study examined the effect of dexamethasone on von Kossa-positive nodule formation and alkaline phosphate specific activity of costochondral chondrocytes at two distinct stages of maturation. The nodules formed by the more mature growth zone chondrocyte cultures contained von Kossa-positive deposits in the extracellular matrix that had a punctate morphology. The nodules formed by the less mature resting zone cells also contained von Kossa-positive deposits, but differentiation was delayed by three-to-five days compared to the growth zone cell cultures. Dexamethasone stimulated the number of nodules formed and shortened the length of time required for von Kossa-positive nodule formation in both types of cultures. During the first 48 h of exposure to dexamethasone, alkaline phosphatase specific activity in the cell layer of both resting zone and growth zone cultures was increased in a dose-dependent manner. At 12 days post-confluence and thereafter, enzyme activity was inhibited in the dexamethasone-treated cultures. Changes in matrix vesicle alkaline phosphatase specific activity reflected those changes seen in the cell layer after dexamethasone treatment, but with higher magnitude, suggesting that one effect of dexamethasone might be to regulate matrix vesicle function. With the exception of one culture, the chondrocytes did not synthesize type X collagen under any of the experimental conditions used. Fourier transform infrared spectroscopy (FTIR) failed to detect the presence of calcium phosphates in any of the cultures exposed to dexamethasone except one. These results demonstrate that dexamethasone promotes early differentiation events, including nodule formation and increased alkaline phosphatase activity, in costochondral chondrocyte cultures. The failure to detect type X collagen synthesis and mineralization in both dexamamethasone-treated and control cultures suggests that these cultures lack the factors necessary for terminal differentiation and mineralization.

Type X collagen in fracture callus and the effects of experimental diabetes.

Studies of fracture repair in diabetes have shown decreased mechanical strength and total collagen in the callus. Type I and Type II collagen are synthesized by bone and cartilage cells, respectively, while Type X collagen is synthesized by hypertrophic chondrocytes in endochondral ossification. A standardized fracture model was chosen to investigate extracellular matrix changes during fracture healing of normal and diabetic rats. Histological examination of the fracture callus in both normal and diabetic animals showed progression from a fibrous tissue to cartilage to bone. An antiserum to Type X collagen was prepared, and immunostaining was observed in the matrix surrounding the hypertrophic chondrocytes. Fracture calluses from streptozotocin induced diabetic rats had similar histology and immunostaining to controls. Radiolabelled proteins were extracted from the calluses of normal and diabetic rats to measure Type X collagen. Type X collagen expression in the fracture callus of normal rats reached a maximum at day fourteen and was decreased by between 54% and 70% in the fracture callus of diabetic rats. The decrease in Type X collagen synthesis may have a role in the defect of fracture healing in diabetes.

Demineralized bone matrix in the stabilization of porous-coated implants in bone defects in rabbits.

Three types of grafts were investigated in rabbits to measure fixation strength of bony ingrowth into porous-coated titanium alloy implants. Autogeneic iliac crest bone (ABG), allogeneic demineralized bone matrix (DBM), and DBM augmented with fibrin glue (DBM + FG) grafts were compared with a press-fit implant control group. Initially, the ABG group required eight weeks and the DBM and DBM + FG groups 12 weeks to achieve fixation strength similar to that of the press-fit group at four weeks. Strength increased with time and at 16 weeks reached 83 kg in the ABG group, 71 kg in the DBM group, and 79 kg in the DBM + FG group, compared with 86 kg for the press-fit group. The ratio of the DBM and the DBM + FG group push-out forces to the ABG push-out force improved from 40% at four weeks to more than 80% at 16 weeks. Histologic analysis of bone ingrowth showed that at 12 weeks, bony ingrowth accounted for 21%, 22%, 16%, and 32% of the porous area in the ABG, DBM, DBM + FG, and press-fit groups, respectively. After eight weeks, there was no statistical difference between DBM, DBM + FG, and ABG grafts in either strength or bony ingrowth. The results demonstrate that over long periods, DBM grafts provide fixation stability comparable with that of autogeneic bone graft.

Two cell lines from bone marrow that differ in terms of collagen synthesis, osteogenic characteristics, and matrix mineralization.

Two cloned cell lines were isolated from cultures of mouse bone-marrow cells. One of the lines, D1, exhibited osteogenic properties and synthesized type-I collagen (alpha 1)2 alpha 2. The second cell line, D2, was not osteogenic and produced a collagen homotrimer (alpha 1)3. Whereas the extracellular matrix of the D1 cell cultures contained striated collagen fibrils, presumably composed of type-I collagen, the homotrimer-producing D2 cells did not demonstrate striated collagen fibrils. Instead, they had thin filaments without detectable striations. Sodium ascorbate stimulated collagen synthesis at the transcriptional level in both the D1 and the D2 cells. The bone-producing characteristics of D1 in vitro included high levels of alkaline phosphatase, increased cyclic adenosine monophosphate on treatment with parathyroid hormone, and expression of osteocalcin mRNA. The D1 cells, unlike the D2 cells, produced a mineralized matrix in vitro. Mineralization in the cultures of the D1 cells occurred in nodules of increased cell density, which also contained the cells with the highest concentrations of collagen mRNA, as shown by in situ hybridization. When the D1 cells were implanted in a diffusion chamber in vivo, a mixture of both osteogenic and adipogenic tissues was formed. This indicates that the D1 cell line is derived from an early marrow stromal precursor that is multipotential.

Matrix mineralization in hypertrophic chondrocyte cultures. Beta glycerophosphate increases type X collagen messenger RNA and the specific activity of pp60c-src kinase.

The phenomenon of chondrocyte hypertrophy is accompanied by the expression of type X collagen and the appearance of matrix mineralization. These events are also associated with changes in the phosphorylation of intracellular proteins. In this study the addition of 10 mM beta-glycerophosphate to hypertrophic chondrocytes resulted in stimulation of type X collagen synthesis up to 10 days in culture and an increase in the expression of type X collagen mRNA. This was followed by the onset of mineralization and the appearance of calcium hydroxyapatite. In contrast, the addition of beta-glycerophosphate to non-hypertrophic chondrocytes failed to induce expression of type X collagen or to produce changes in calcium and phosphate. The increased formation of type X collagen and of mineral in hypertrophic chondrocytes was accompanied by changes in the tyrosine kinase pp60c-src. While the level of c-src protein decreased approximately 2.5-fold in hypertrophic chondrocytes after 17 days of beta-glycerophosphate treatment, the specific activity of pp60c-src kinase increased approximately 3-fold in the cells that could be induced to mineralize but remained unchanged in cells that did not exhibit this property. Regulation of kinase activity may be an important event in endochondral ossification.

A 17-kd polypeptide, sensitive to bacterial collagenase, is synthesized by bone marrow macrophages.

A novel polypeptide with a molecular weight of 17 kd (17k protein) was identified in bone marrow cell cultures. The synthesis of 17k protein is elevated in cell cultures maintained under Dexter conditions, which support myelopoiesis. The predominance of macrophages in the stromal layer of these cultures and the observation that a mouse myelomonocytic cell line P388D1 is capable of synthesizing large amounts of 17k protein led us to the study of its synthesis by bone marrow macrophages. Metabolic labeling with [14C]proline and partial amino acid analysis of 17k protein demonstrated that the polypeptide contains relatively high amounts of proline and is also sensitive to degradation with bacterial collagenase. However, no hydroxyproline is detectable in 17k protein, and it is extensively degraded with bacterial collagenase. However, no hydroxyproline is detectable in 17k protein, and it is extensively degraded by proteolysis with pepsin, using conditions under which collagen triple helices are resistant to degradation, suggesting that collagen-like structures are not contained in 17k protein. This polypeptide is found predominantly in the cellular layers of bone marrow macrophage cultures. Incorporation of [14C]proline into 17k protein is diminished by increasing concentrations of colony-stimulating factor 1 (CSF-1). The 17k protein may be involved in macrophage proliferation because its synthesis is inhibited by CSF-1, which is required for the maintenance of bone marrow macrophages in vitro.