The use of bone-graft substitutes in large bone defects: any specific needs?

INTRODUCTION: The gold standard for restoring bone defects is still considered to be autologous bone grafting. However, clinical benefits are not guaranteed and donor-site complications and morbidity is not infrequent. Research is on-going for the development of alternative bone substitutes of both biological and synthetic origin. The purpose of this study was to evaluate the type of materials used and their efficacy for the treatment of large bone defects in traumatology and orthopaedic surgery. MATERIALS AND METHOD: A literature review was carried out of Embase and PubMed databases. Inclusion criteria were articles in English language focusing on the use of bone substitutes in trauma and orthopaedic surgery for the treatment of bone defects and included details on the structural, biological or biomechanical properties of the pure product. Furthermore, based on two clinical challenges, fracture non-union and impaction grafting we elaborated on the use of polytherapy for large bone defects as guided by the diamond concept. RESULTS: All the products indicated in this manuscript possess osteoconductive activities but have different resorption times and biomechanical properties. Bone graft substitute materials are used for a wide range of clinical applications even when the level of clinical evidence is low. The size and location of the defect and the local biological and mechanical environment as well as the biomechanical characteristics of the material determine the type of device that can be implanted in a bone defect. CONCLUSION: Proper assessment of the biological and mechanical environment and accurate patient selection are necessary to judge the extent of therapy the injury warrants. A sound understanding of various aspects of biomaterial properties and their relation and influence towards bone healing is of utmost importance. We suggest the application of polytherapy for the treatment of large bone defects and advocate the use of the diamond concept as a guideline.

Bisphosphonate treatment modifies canine bone mineral and matrix properties and their heterogeneity.

Bone loss and alterations in bone quality are major causes leading to bone fragility in postmenopausal women. Although bisphosphonates are well known to reduce bone turnover and prevent bone loss in postmenopausal osteoporosis, their effects on other bone properties are not fully characterized. Changes in bone mineral and matrix properties may contribute to the anti-fracture efficacy observed with bisphosphonate treatments. The aim of this work was to analyze the effect of a 1-year treatment with either alendronate or risedronate, at low and high doses, on spatially resolved bone material and compositional properties that could contribute to the fracture efficacy of these agents. Distal tibias from 30 normal beagles that had been treated daily for 1 year with oral doses of vehicle (Veh), alendronate (Aln) at 0.2 or 1 mg/kg, and risedronate (Ris) at 0.1 or 0.5 mg/kg were analyzed by Fourier Transform Infrared imaging (FTIRI) to assess the changes in both mineral and matrix properties in discrete bone areas. The widths at half maximum of the pixel histograms for each FTIRI parameter were used to assess the heterogeneity of the bone tissue. Aln and Ris increased the mineral content and the collagen maturity mainly in cancellous bone and at the endocortical surface. Significant differences were observed in the mineral content and in the hydroxyapatite crystallinity distribution in bone tissue, which can contribute to reduced ductility and micro-crack accumulation. No significant differences were observed between low and high dose nor between Aln and Ris treatments. These results show that pharmacologic suppression of bone turnover increases the mineral and matrix bone tissue maturity in normal cancellous and endocortical bone areas where bone turnover is higher. These positive effects for decreased fracture risk are also associated with a loss of bone heterogeneity that could be one factor contributing to increased bone tissue brittleness and micro-crack accumulation.

Effect of hyperbaric oxygen on demineralized bone matrix and biphasic calcium phosphate bone substitutes.

OBJECTIVES: The aim of this study was to assess the possible effect of hyperbaric oxygen (HBO) on the healing of critical-sized defects that were grafted with demineralized bone matrix (DBM) combined with Pluronic F127 (F127) to form a gel or putty, or a commercially available biphasic calcium phosphate (BCP), mixed either with blood or F127 to form a putty. STUDY DESIGN: Twenty New Zealand White rabbits were randomly divided into 2 groups of 10 animals each. Bilateral 15-mm calvarial defects were created in the parietal bones of each animal, resulting in 40 critical-sized defects. Group I defects were grafted with either DBM putty or DBM gel. Group II defects were grafted with either BCP or BCP putty. Five animals from each group received HBO treatment (100% oxygen, at 2.4 ATA) for 90 minutes per day 5 days a week for 4 weeks. The other 5 animals in each group served as a normobaric (NBO) controls, breathing only room air. All animals were humanely killed at 6 weeks. The calvariae were removed and analyzed by micro computed tomography (mCT) and histomorphometry. RESULTS: mCT analysis indicated a higher bone mineral content (BMC, P < .05), bone volume fraction (BVF; P < .001), and bone mineral density (BMD; P < .001) of the defects grafted with BCP rather than DBM. Furthermore, the voxels that were counted as bone had a higher tissue mineral density (TMD) in the BCP- than in the DBM-filled defects (P < .001). Histologically complete bony union over the defects was observed in all specimens. Histomorphometric analysis showed that DBM-filled defects had more new bone (P < .007) and marrow (P < .001), and reduced fibrous tissue compared with the BCP defects (P < .001) under NBO conditions. HBO treatment reduced the amount of fibrous tissue in BCP filled defects (P < .05), approaching levels similar to that in matching DBM-filled defects. HBO also resulted in a small but significant increase in new bone in DBM-grafted defects (P < .05). CONCLUSION: Use of DBM or BCP promoted healing in these critical-sized defects. Hyperbaric oxygen therapy resulted in a slight increase in new bone in DBM-grafted defects and much larger reduction in fibrous tissue and matching increases in marrow in BCP-grafted defects, possibly through increased promotion of angiogenesis.

[Role of enamel organic matrix in the remineralization of initial demineralized enamel and artificial hydroxylapatite treated with Galla chinensis].

OBJECTIVE: To observe the remineralization of initial demineralized enamel and artificial hydroxylapatite treated with Galla chinensis in vitro, and to assess the effect of enamel organic matrix on the potential of Galla chinensis to promote the remineralization of initial enamel carious lesions, further to elucidate the mechanism of Galla chinensis in promoting the remineralization of initial enamel carious lesion. METHODS: Bovine sound enamel blocks, non-organic enamel blocks and artificial hydroxylapatite blocks were demineralized and exposed to a pH-cycling. During the pH-cycling, the specimens were randomly treated with 1 g/L NaF, 4 g/L Galla chinensis extract (GCE) or double deionized water (DDW). Surface microhardness of all the samples was measured before and after the pH-cycling, and percentage surface microhardness recovery (% SMHR) was calculated. The surface morphology was observed by scanning electron microscopy. RESULTS: A significant increase in microhardness (P < 0.05) with many irregular deposits and prominences on GCE treated regular enamel blocks were observed. No significant increase in that of the regular enamel treated with DDW, enamel disposed of its organic matrix or artificial hydroxylapatite treated with GCE were observed (P > 0.05). No obvious changes in the SEM images of regular enamel treated with DDW, enamel disposed of its organic matrix or artificial hydroxylapatite treated with GCE compared to those of them before pH-cycling. CONCLUSION: Galla chinensis enhances the remineralization of initial enamel carious lesions in vitro. The organic matrix of enamel was shown to play a substantial role in the observed mechanism.

The effect of thrombin activation of platelet-rich plasma on demineralized bone matrix osteoinductivity.

BACKGROUND: Demineralized bone matrix is an osteoinductive and osteoconductive material that is often used in orthopaedic surgery to induce bone formation. Autologous platelet-rich plasma, which contains proliferative and chemoattractant growth factors, has been used as a demineralized bone matrix adjuvant with mixed results. One variable during clinical use appears to be whether the platelet-rich plasma is activated with thrombin or is implanted in a liquid form with intact platelets. The objective of the present study was to determine if platelet-rich plasma can increase the osteoinductivity of demineralized bone matrix when used without thrombin activation. METHODS: The bioactivity of the demineralized bone matrix was evaluated in vitro by determining alkaline phosphatase production by C2C12 myoblast cells. The effect of thrombin activation on platelet-rich plasma was studied in vitro by evaluating osteosarcoma and bone marrow stromal cells for cell number and transforming growth factor-beta1 activation. Demineralized bone matrices supplemented with platelet-rich plasma, with or without thrombin activation, were implanted intramuscularly in athymic rats and were examined at fourteen, twenty-eight, and fifty-six days. Histological samples were analyzed for osteogenesis and chondrogenesis. Osteogenesis was further characterized on the basis of alkaline phosphatase activity. RESULTS: In vitro, thrombin triggered an immediate release of growth factors from the platelet-rich plasma, and the platelet-rich plasma increased the number of both osteosarcoma and stromal cells in a dose-dependent manner. Thrombin activation of the platelet-rich plasma eliminated such stimulatory effects. In vivo, the platelet-rich plasma stimulated chondrogenesis on Day 14 and osteogenesis on Days 28 and 56, whereas thrombin-activated platelet-rich plasma acted as an inhibitor of such events. In addition, inflammatory cells were detected in demineralized bone matrix samples that were mixed with thrombin-activated platelet-rich plasma. These cells were not present in matrix mixed with platelet-rich plasma alone. CONCLUSIONS: Platelet-rich plasma significantly increased in vivo demineralized bone matrix osteoinductivity only when used without thrombin activation.

Characterisation of the constitutive over-expression of AJ18 in a novel rat stromal bone marrow cell line (D8-SBMC).

OBJECTIVE: The elucidation of the molecular pathways involved in osteoblast proliferation and differentiation has been greatly enhanced by the availability of cell culture model systems. However, many of the current bone cell culture systems suffer from disadvantages such as the inability to generate mineralised bone-like nodules, a transformed genetic background, cell heterogeneity, and a relatively long time frame from cell seeding to mineralisation, often in the order of several weeks. Here we describe the establishment and characterisation of a novel bone cell line named D8-SBMC. As a first demonstration of their potential value, D8-SBMC was utilised to further support a role for AJ18 during osteogenesis. DESIGN: D8-SBMC was established from a single cell suspension of the previously characterised long term rat stromal bone marrow cells [Kotev-Emeth S, Pitaru S, Pri-Chen S, Savion N. Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2. Connect Tissue Res 2002;43(4):606-12; Pitaru S, Kotev-Emeth S, Noff D, Kaffuler S, Savion N. Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture. J Bone Miner Res 1993;8(8):919-29]. AJ18 was constitutively and stably over-expressed in D8-SBMC and analysed. RESULTS: D8-SBMC possesses the ability to form robust mineralised bone-like nodules within 8 days proceeding cell confluency. Interestingly, a cement line-like matrix is also generated between the culture dish and a basal monolayer of cells. Constitutive and stable over-expression of AJ18 resulted in an increase in cell proliferation and mineralisation. Expression of bone marker genes, such as bone sialoprotein, osteopontin, osteocalcin, collage type 1, and osteonectin, was up-regulated by AJ18 over-expression. CONCLUSION: A novel bone cell line, D8-SBMC, was established and characterised. D8-SBMC may be a valuable model system for biomineralisation studies. D8-SBMC was utilised to further understand the role of AJ18 in cell proliferation and differentiation during osteogenesis.

Bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro.

Bone matrix production and mineralization involves sophisticated mechanisms, including the initial formation of an organic extracellular matrix into which inorganic hydroxyapatite crystals are later deposited. Human embryonic stem (hES) cells offer a potential to study early developmental processes and provide an unlimited source of cells. In this study, four different hES cell lines were used, and two different approaches to differentiate hES cells into the osteogenic lineage were taken. Undifferentiated cells were cultured either in suspension, facilitating the formation of embryoid bodies (EBs), or in monolayer, and both methods were in the presence of osteogenic supplements. Novel to our osteogenic differentiation study was the use of commercially available human foreskin fibroblasts to support the undifferentiated growth of the hES cell colonies, and their propagation in serum replacement-containing medium. Characterization of the osteogenic phenotype revealed that all hES cell lines differentiated toward the mesenchymal lineage, because T-Brachyury, Flt-1, and bone morphogenetic protein-4 could be detected. Main osteoblastic marker genes Runx2, osterix, bone sialoprotein, and osteocalcin were up-regulated. Alizarin Red S staining demonstrated the formation of bone-like nodules, and bone sialoprotein and osteocalcin were localized to these foci by immunohistochemistry. Cells differentiated in monolayer conditions exhibited greater osteogenic potential compared to those from EB-derived cells. We conclude that in vitro hES cells can produce a mineralized matrix possessing all the major bone markers, the differentiation of pluripotent hES cells to an osteogenic lineage does not require initiation via EB formation, and that lineage potential is not dependent on the mode of differentiation induction but on a cell line itself.

Augmented bone-matrix formation and osteogenesis under magnetic field stimulation in vivo XRD, TEM and SEM investigations.

Bone is a composite biomaterial, which is formed, when proteins constituting collagen fibers attract calcium, phosphate and hydroxide ions in solution to nucleate atop the fibers. It grows into a hard structure of tiny crystallites of hydroxyapatite, aligned along the long axis of collagen fibers. The present work reports the stimulating effect of static magnetic field on microstructure and mineralization process of bone repair. A unilateral transverse fracture of mid-shaft of metacarpal was surgically created in healthy goats under thiopental sedation and xylocaine analgesia. Two bar magnets (approximately 800 gauss/cm2 field strength) were placed across the fracture line at opposite pole alignment immobilized in Plaster of Paris (POP) splint bandage for static magnetic field stimulation. Radiographs were taken at weekly intervals up to 45 days. Results show that formation of extra-cellular matrix and its microstructure can be influenced by non-invasive physical stimulus (magnetic field) for achieving an enhanced osteogenesis, leading to quicker regeneration of bone tissue in goats. X-ray diffraction (XRD) patterns of treated (magnetic field-exposed) and control samples revealed the presence and orientation of crystalline structures. Intensity of diffraction peaks corresponding to 310 and 222 planes were enhanced with respect to 211 families of reflections, indicating preferential alignment of the crystals. Also, the percent crystallinity and crystal size were increased in treated samples. The study provides a biophysical basis for augmented fracture healing under the influence of semi-aligned static magnetic field applied across the fracture line.

Assessment of bone mineral and matrix using backscatter electron imaging and FTIR imaging.

The resistance of bone to fracture is determined by its geometric and material properties. The geometry and density can be determined by radiographic methods, but material properties such as collagen structure, mineral composition, and crystal structure currently require analysis by invasive techniques. Backscatter electron imaging provides quantitative information on the distribution of the mineral within tissue sections, and infrared and other vibrational spectroscopic methods can supplement these data, providing site-specific information on mineral content as well as information on collagen maturity and distributions of crystal size and composition. This information contributes to the knowledge of "bone quality."

In vitro re-mineralization of demineralized bone matrix in human serum.

The aim of this study was to develop an in vitro re-mineralization model in human serum. For this purpose a commercially produced demineralized human bone matrix (DBM) was incubated in samples of human serum pools obtained from two physiologically different groups. The first group consisted of young males and the second of older females. After incubation periods of 4 and 7 days at 37 degrees C, changes in the levels of calcium and inorganic phosphate content of the serum and DBM samples were measured. The results of the study showed that the change in mineral content of serum and DBM samples in both study groups was statistically significant. The decrease in serum calcium content and increase in DBM inorganic phosphate content were significant in the young group for longer incubation times. In the older group, both serum calcium and inorganic phosphate decreased and DBM mineral content increased for the same incubation time. When the two physiological groups were compared, statistically significant differences were identified for changes in mineral levels in both serum and the DBM samples. These data indicate that the mineral content of human serum decreases and that of DBM increases when these two materials are incubated together. These changes provide evidence for the re-mineralization of DBM. The model described here could also detect a difference in re-mineralization capability between two different groups of human sera.

Exogenous Runx2 expression enhances in vitro osteoblastic differentiation and mineralization in primary bone marrow stromal cells.

Bone marrow stromal cells represent a promising cell source for cell-based therapeutic and bone tissue-engineering applications, but are restricted by a low frequency in healthy marrow, an age-related decrease in osteogenic capacity, and a propensity for dedifferentiation during in vitro expansion. To address these limitations, retroviral gene delivery was used to examine the effects of sustained and elevated expression of the Runx2 osteoblastic transcription factor on osteoblastic gene and protein expression and mineralization in primary rat bone marrow stromal cells. Runx2 overexpression upregulated several osteoblast-specific genes, including collagen type I and osteocalcin, and enhanced alkaline phosphatase activity and biological mineral deposition. Forced Runx2 expression in combination with dexamethasone increased matrix mineralization compared with exogenous Runx2 expression or dexamethasone treatment alone, whereas dexamethasone-free control cultures displayed minimal mineralization. These additive effects suggest complementary interactions between Runx2 and dexamethasone-responsive regulatory factors. Finally, Runx2 overexpression in stromal cell cultures undergoing considerable in vitro expansion resulted in higher matrix mineralization capacity compared with controls, which completely lost the ability to produce mineralized matrix even in the presence of dexamethasone. These findings provide a novel strategy for cell-based therapeutic applications requiring significant numbers of osteogenic cells to synthesize mineralized constructs for the treatment of large bone defects.

Suppressed bone induction by follistatin in spontaneously hypercholesterolemic rat bone.

Bone inducing activity in demineralized bone matrix (DBM) of young spontaneously hypercholesterolemic (SHC) rats has been shown to be lower than that of aged SHC rats. This study examined the involvement of bone follistatin, an activin-binding protein, in bone induction. Immunoreactive follistatin was higher in DBM from 10-week-old SHC rats (DBM-10wk) than in DBM from 6-month-old SHC rats (DBM-6mo). When DBM without follistatin supplement was implanted, the C-propeptide of type II procollagen and calcium contents on day 12 in implants of DBM-6mo were 68% and 40% higher than those of DBM-10wk, respectively. In contrast, follistatin supplement to DBM decreased C-propeptide of type II procollagen and calcium contents in implants of both DBM-10wk and DBM-6mo, and the levels of these parameters were comparable between DBM-10wk and DBM-6mo, indicating reduced formation of cartilage and bone. These findings suggest that 1) follistatin content in bone matrix decreases with advancing age in SHC rats, and 2) the follistatin interferes with endochondral bone formation. We demonstrate that the lower bone induction of DBM from young SHC rats was partly due to the abundance of follistatin in bone matrix.

Histologic study of the effect of hyperbaric oxygen therapy on autogenous free bone grafts.

PURPOSE: This study was undertaken to evaluate the effect of hyperbaric oxygen therapy on autogenous free bone grafts transplanted from iliac crest to the mandibles of rabbits. MATERIALS AND METHODS: A piece of corticocancellous bone harvested from iliac crest was grafted to a same-size bone defect in the mandibles of 16 Japanese white rabbits. In 8 rabbits, 20 and 10 sessions of hyperbaric oxygen (HBO) treatment were carried out twice per day (2.4 ATA 60 min.) before and after operation, respectively. The other 8 rabbits served as controls. The graft and surrounding bone were sampled at 1, 2, 4, or 8 weeks after transplantation, and the effects of HBO were evaluated by light micrography and contact microradiography. RESULTS: At 1 week after grafting, osteoid formation in the experimental group was much greater than in the control group. Union between the grafted and the host bone was observed in the experimental group at 2 weeks after grafting, but it was not observed in the control group until 4 weeks. Although it was difficult to differentiate grafted from host bone in the experimental group at 4 weeks, it was readily distinguishable in the control group. CONCLUSIONS: These results indicate that HBO accelerates the union of autogenous free bone grafts.

Evaluation of distraction osteogenesis by scanning electron microscopy.

A model of bifocal distraction osteogenesis in the canine model was used to assess and quantitate the mineral content of the newly forming bone within the canine mandible. A 2-cm defect was created in the body of the mandible, and after a posterior osteotomy, the transport disk was advanced at 0.25 mm per 8 hours for 21 days and then held in rigid fixation for an additional week. As a control for this study, three additional dogs underwent the same procedure with the exception that the transport disk was not advanced. Electron dispersive spectroscopy analysis was performed on the newly formed regenerate bone and compared with areas of existing cortical bone of both the transport disk and the mandible. In the control model, special note was made of the pericortical callus at the osteotomy site as well as of the regenerative bone that filled the 2-cm defect in the body of the mandible. Calcium/phosphorous ratios were used to assess the composition of the mineralized regions of the mandible. The regenerate bone that filled the defect and the mineralized callus surrounding the site of osteoclasis in the control mandible were significantly different in composition when compared with the regenerate bone that formed during distraction osteogenesis. This suggests that distraction osteogenesis may effect an initial matrix production that is more similar in composition to the mature cortical bone from which it was derived than does periosteal regeneration and filling of an osseous defect.

Osteogenesis associated with bone gla protein gene expression in diffusion chambers by bone marrow cells with demineralized bone matrix.

Diffusion chambers with rat bone marrow cells and demineralized bone matrix (DBM) were implanted subcutaneously to syngeneic 8-week-old rats and were harvested every week 3-7 weeks after implantation, and histochemical examination, determination of alkaline phosphatase activity, total calcium and phosphorus, the bone-specific vitamin K-dependent gla-containing protein (BGP) content, and detection of BGP mRNA relative to mineralization were performed. Alkaline phosphatase in diffusion chamber implants reached the highest activity at 4 weeks and then decreased. Calcium and phosphorus deposits occurred at 4 weeks after implantation and were followed by marked increases until 7 weeks, which was comparable to the accumulation of BGP. The BGP gene within the diffusion chambers began to be expressed at 5 weeks, and its expression increased markedly at 7 weeks after implantation. At 4-5 weeks after implantation, new bone adjacent to the membrane filters and cartilage toward the center of the diffusion chamber were observed histochemically. Light microscopic and immunohistologic examinations of chambers with marrow cells and DBM revealed production of mineralized matrices, typical of bone characterized by the appearance of BGP and mineralized nodules. In contrast, bone marrow cells alone did not show extensive bone formation and yielded very low values for these biochemical parameters. The present experiments demonstrate the potential of bone marrow cells and DBM to produce not only cartilage formation but also membranous bone formation associated with increasing expression of BGP mRNA during the later stages of bone formation, as well as a marked accumulation of BGP.

Influence of parathyroidectomy, 1,25-dihydroxyvitamin D3 and high dietary calcium intake on demineralized bone matrix powder-induced bone formation in the rat.

Demineralized bone matrix induces ectopic endochondral bone formation. We used this model to study the effect of parathyroidectomy (PTX), 1,25-dihydroxyvitamin D3 treatment, and calcium enriched diet on bone formation in the rat. Hypocalcemia and hyperphosphatemia in PTX rats were corrected by 1,25-dihydroxyvitamin D3 treatment (2 x 12.5 ng/day) or by calcium enriched diet (3% calcium). Serum 1,25-dihydroxyvitamin D3 concentration was decreased in PTX rats and in intact rats with high dietary calcium intake. Calcium content of ectopic new bones (42 days after bone matrix implantation) was reduced in PTX rats compared with intact control rats. This could be prevented by 1,25-dihydroxyvitamin D3 treatment. In contrast, calcium enriched diet led to diminished mineralization of ectopic bones both in intact and PTX rats. We conclude that the effect of parathyroidectomy on bone formation may be mediated by 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 directly stimulates bone formation in this model and this effect is not simply the result of increasing serum calcium concentration.

Ectopic induction of cartilage and bone by water-soluble proteins from bovine bone using a collagenous delivery vehicle.

A controlled-release delivery vehicle for water-soluble osteogenic proteins from demineralized bone matrix was constructed using purified type I collagen. The water-soluble proteins were isolated from a 4 M GdnHCl extract of bone matrix. Although the water-soluble proteins were capable of inducing cartilage formation in vitro, they were incapable of inducing cartilage or bone in vivo when implanted intramuscularly into mice in the absence of an appropriate delivery vehicle. The collagen-based delivery vehicle alone was also incapable of inducing osteogenesis in vivo. However, when the water-soluble proteins were incorporated into the delivery vehicle, the combination was capable of inducing cartilage and bone 76% of the time. These results demonstrate that it is possible to formulate a controlled-release delivery vehicles for soluble bioactive factors which upon release interact with local responsive cells.

[Changes in the osteoinductive activity of the bone matrix in ontogeny]. / Izmenenie osteoinduktivnoi aktivnosti kostnogo matriksa v ontogeneze.

Correlation between the animal's age, degree of mineralization of its bone tissue and the osteoinductive activity of the bone organic matrix was established in experiments with rats of the same litter at an age of 2, 4, 8, 16 weeks. The osteoinductive activity was estimated by the capacity of matrix to induce ectopic osteogenesis using biochemical methods. Bone mineralization increased and the capacity for osteoinduction decreased roughly 1,5-fold with the age of donor animals. It is suggested that the reduction of osteoinductive potencies is based on the decrease in lability of the bond between the protein osteoinducer and the collagen matrix, as shown by unequal sensitivity of bone matrix of rats of different age to the doubling of the shortest possible time of bone demineralization.

Platelet-derived growth factor enhances demineralized bone matrix-induced cartilage and bone formation.

Subcutaneous implantation of demineralized bone matrix induces the local formation of cartilage and bone. In this study we have investigated the influence of adding various growth factors to the implant. Cartilage formation was monitored by measuring collagen II mRNA levels, and bone formation in the implant was assessed from alkaline phosphatase activity and calcium content. Supplements of the platelet-derived growth factor to implants in older rats increased and production of mRNA for collagen II, alkaline phosphatase activity, and the calcium content of the implant, whereas the other growth factors tested were without effect. The data suggest that under some conditions bone induction is submaximal and can be increased by local supplement of platelet-derived growth factor (PDGF). The present observations may have important therapeutic implications in the treatment of nonunions of fractures and impaired bone formation in the aged.

The effect of decalcified bone matrix on the osteogenic potential of bone marrow.

Rabbit bone marrow cells were cultured in diffusion chambers with or without decalcified bone matrix. The chambers were assayed after 28 days for alkaline phosphatase activity, deoxyribonucleic acid (DNA), calcium, and phosphorus contents. Morphologically, marrow cells incubated with or without matrix differentiated to form bone and cartilage. With bone matrix, the calcium and phosphorus contents of chambers were significantly higher than control chambers. Alkaline phosphatase activity and DNA content were not influenced by inclusion of bone matrix. These results indicate that bone matrix constituents exert a stimulatory effect on bone formation from marrow cells. This osteogenic stimulation could be due to the influence of an osteoinductive factor and/or to stimulation of osteoprogenitor cells known to be present in the marrow.