Bone Matrix Particles Research Articles

Maxillary sinus floor augmentation on humans: Packing simulations and 8 months histomorphometric comparative study of anorganic bone matrix and β-tricalcium phosphate particles as grafting materials

The present study compares the behaviour of an anorganic bone matrix material and a synthetic β-Tricalcium phosphate employed as grafting materials in a sinus floor augmentation two step protocol in humans. In order to estimate the initial occupation level for the two materials, an ‘in vitro’ simulation has been performed to analyse macroporosity created due to particle packing in terms of porosity and interparticle distances. Grafting in the sinus floor augmentation was performed by filling the defects only with pure grafting materials without autogenous bone addition. The new-bone generated is 100% based on the osteoconductive properties of the grafted materials in contact with physiological fluids. The implants were placed 8 months after the grafting procedure. All the implanted positions were biopsied and embedded in methacrylate resin. Histomorphometric analyses were done over thin film undecalcified sections. Packing simulations allow establishing a comparison of the resorbed volumes related to the initial occupancy of the grafting materials inside the defect. The nature of this interconnected pore network is very alike for either material so new-bone generated was similar (∼ 35 vol.%).

Combination of vascularized outer-table calvarial bone graft based on the superficial temporal vessels and allomatrix for the repair of an orbito-frontal blow-out fracture in a child

A vascularized outer-table calvarial bone graft was used for repairing a Posnick type 2 traumatic orbito-frontal bone defect supported by the use of a calcium-based putty (Allomatrix) in a 7-year-old girl. Gaps between the donor and recipient sites were filled with Allomatrix containing demineralized bone matrix particles. Four years later there was a good cosmetic result using an artificial left eye. Orbito-frontal defects have been repaired using iso-, allo- or xenografts as well as synthetic materials. Anatomical studies have pointed to the importance of the superficial temporal vessels for the vascular supply of a graft to the calvaria. The vascularized outer-table calvarial bone graft based on the superficial temporal vessels is a reliable option for repairing bony defects in the craniofacial skeleton especially in irradiated or scarred areas where there is a paucity of well-vascularized tissues. It also has the potential for bony growth in children.

Rapid quantitative bioassay of osteoinduction.

We developed a reproducible, relatively rapid bioassay that quantitatively correlates with the osteoinductive capacity of demineralized bone matrix obtained from human long bones. We have found that Saos human osteosarcoma cells proliferate in response to incubation with demineralized bone matrix and that an index of this proliferative activity correlates with demineralized bone matrix-induced osteogenesis in vivo. The bioassay (Saos cell proliferation) had an interassay coefficient of variation of 23 +/- 2% and an intra-assay coefficient of 11 +/- 1%. Cell proliferation was normalized to a standard sample of demineralized bone matrix with a clinically high osteoinductive capacity, which was assigned a value of one. The Saos cell proliferation for each sample was related to the standard and assigned a value placing it into the low (0.00-0.39), intermediate (0.40-0.69), or high (0.70-1.49) osteoinductive index group. Osteoinduction of human demineralized bone matrix was quantitated by expressing new bone formation as a function of the total bone volume (new bone plus the demineralized bone powder). The demineralized bone matrix was placed in pouches formed in the rectus abdominis muscles of athymic rats, and endochondral bone formation was assessed at 35 days following implantation, when marrow spaces in the ossicles were formed by new bone bridging the spaces between demineralized bone matrix particles. The proliferative index correlated with the area of new bone formation in histological sections of the newly formed ossicles. When the proliferative index (the osteoinductive index) was divided into low, intermediate, and high groups, the correlation between it and new bone formation (osteoinduction) was 0.850 (p < 0.0005) in 25 samples of demineralized bone matrix. There was no overlap in the osteoinduction stimulated between the samples with low and high osteoinductive indices. We conclude that the proliferation assay is useful for the routine screening of bone allograft donors for osteoinductive potential. Furthermore, the two-dimensional area of new bone formation, as it relates to total new bone area, is a quantitative measure of osteoinduction.

Inhibition of matrix-induced bone differentiation by advanced glycation end-products in rats.

Glycation of long-lived proteins is an inevitable consequence of aging that is accelerated in patients with diabetes mellitus. Treatment of demineralized bone matrix particles from 35-week-old normal Long-Evans rats with glycoaldehyde, a precursor of advanced glycation end-products, was used to assess the effects of bone-matrix glycation on the process of bone differentiation. Matrix was incubated in phosphate buffered saline alone, phosphate buffered saline containing glycolaldehyde, glycolaldehyde plus the advanced glycation product-inhibitor aminoguanidine, or glycolaldehyde plus the advanced glycation product-inhibitor sodium cyanoborohydride. Glycolaldehyde increased the matrix advanced glycation product content as measured by specific fluorescence more than two-fold, while inhibiting bone differentiation more than 90% as measured by in vivo 45CaCl2 uptake, alkaline phosphatase levels, and histology. In contrast, simultaneous incubation with the advanced glycation product-inhibitor aminoguanidine or sodium cyanoborohydride not only reduced fluorescence to normal, but also restored bone differentiation. Furthermore, the inhibition of bone differentiation by glycolaldehyde was not reversed by subsequent application of recombinant bone morphogenetic protein-2. These observations suggest that formation of advanced glycation products on bone matrix alters its ability to induce bone formation, and probably involves alterations of binding sites for extractable proteins with direct bone inductive properties such as bone morphogenetic protein-2. Decreased bone formation associated with aging and diabetes may result, in part, from advanced glycation product formation on matrix proteins.

The functional equivalence of demineralized bone and tooth matrices in ectopic bone induction.

The objective of this study was to determine whether demineralized rat incisor matrices were a more potent inducer of ectopic endochondral bone formation than demineralized diaphyseal bone matrices derived from the same donors. Twenty-five-milligram disks of demineralized bone or tooth matrix obtained from adolescent Long-Evans rats were implanted in a standardized ectopic site. Biochemical and histometric measurements of bone formation revealed that the two matrices were functionally equivalent inducers of endochondral bone formation. The induced pellicle of bone reached a maturation point 18 days after implantation. Dentin matrix implants generated a significantly greater amount of mineralized tissue than did bone matrix implants. This difference could be explained on the basis of remineralization of the dentin particles to a greater degree than the bone matrix particles. Initial observations suggesting a more robust osteoinductive activity in demineralized incisor matrix can be attributed to the decreasing activity of bone matrix from older donors when compared to younger donors. The extent of osteoinduction by the two substrata was equivalent when the matrices were matched for age.

Accelerated endochondral osteoinduction in the absence of bone matrix particles in a rat model system

Ethanol-precipitated proteins obtained from demineralized rat bone powder (DBP) by 4M guanidine-HCl extraction have been shown to reproducibly induce ectopic endochondral bone formation when subcutaneously implanted in rats in the absence of bone matrix particles. Histologic and biochemical analysis revealed a temporal sequence of chondrocyte differentiation, calcified cartilage formation, neovascularization, osteoblast differentiation, bone formation, osteoclastic bone remodeling, and hematopoietic marrow development that is complete by 21 days. In contrast to previous reports, these results clearly show an osteoinductive response independent of the presence of insoluble extracellular bone matrix. Compared with conventional DBP implants, the guanidine-extractable protein (GE) produces an accelerated and more robust osteoinductive response. Histologically, the initial chondrogenic response at days 6 to 9 is greatly amplified. Alkaline phosphatase specific activity peaks at day 9, several days earlier than for DBP, and is sixfold higher. Calcium accumulation in GE implants at day 12 is fivefold greater than with DBP, and all mineral is localized within the matrix of newly calcified cartilage and new bone. Osteoclasts are up to ninefold more abundant in the rapidly remodeling GE ossicle, making space for hematopoietic marrow. Delivery of GE coprecipitated with inert bone matrix particles was also more effective than DBP, although the response was somewhat attenuated compared with GE alone. Bony filling of 4-mm defects in rat mandibular rami was elicited by 10 mg of GE and followed an endochondral process with increased neovascularization compared with DBP and unimplanted controls. This guanidine-extractable protein fraction should prove useful for inducing quantities of chondrocytes and osteoclasts for in vitro study, and for analysis of osteoinductive requirements. Likewise, ethanol-precipitated osteoinductive proteins implanted alone or with carrier particles hold promise for use in reconstructive surgery.

Discussion: Accelerated Endochondral Osteoinduction in the Absence of Bone Matrix Particles in a Rat Model System

Discussion: Accelerated Endochondral Osteoinduction in the Absence of Bone Matrix Particles in a Rat Model System

Bone induction by composite of bioerodible polyorthoester and demineralized bone matrix in rats

A composite of a local, sustained, drug-release system, Alzamer bioerodible polyorthoester, and demineralized bone-matrix (DBM) particles implanted in the abdominal muscle of 89 Wistar rats induced cartilage and bone formation at the same rate as DBM when evaluated histologically and by 85Sr uptake. The composite implant was technically easier to use than DBM alone.

Decalcified tooth matrix powder induces new bone formation and hematopoietic microenvironment in the mouse.

Implants of bone and tooth matrix powder were placed subcutaneously (s.c.) on intraperitoneal (i.p.) Mitex or Polyvic membranes. Implants were removed for histology after 1-24 weeks. Macrophages, fibroblasts, and vascular sinusoids infiltrated around bone and tooth matrix particles after one week. In the s.c. tooth matrix implants, a few sites of cartilage formation and ossification developed at two weeks, and by three weeks granulocytopoiesis and megakaryocytopoiesis developed adjacent to new bone; erythropoiesis was not observed. In s.c. bone matrix implants and in the i.p. artificial membranes coated with bone or tooth matrix powder, ossification or hematopoiesis was not observed. Small numbers of CFU-s, CFU-nm, BFU-e, and CFU-e appeared 10-20 days after s.c. implantation of tooth matrix; none were detected in s.c. bone matrix implants.

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.