An evaluation of human demineralized bone matrices in a rat femoral defect model

The osteoconductive and osteoinductive potential of two human allogeneic demineralized bone matrix putties were compared in a critical-sized athymic rat femoral defect model. Defects were treated with (1) a demineralized bone matrix in a hyaluronic acid carrier, (2) a demineralized bone matrix in a glycerol carrier, (3) a hyaluronic acid carrier alone, or (4) with no implant. Radiographic examinations and histologic analyses were done at 4, 8, and 16 weeks postoperatively. Eight of the 48 defects treated with a demineralized bone matrix and none of the 36 surgical controls showed complete radiographic healing by 16 weeks and no statistically significant difference between the radiographic scores for the two demineralized bone matrix preparations was found. On histologic review, both preparations of demineralized bone matrix had passive remineralization. The largest foci of endochondral ossification were seen in limbs treated with a demineralized bone matrix in a hyaluronic acid carrier. The 8-mm rat femoral defect allows for stringent assessment of the osteoinductive potential of bone graft substitutes. Hyaluronic acid and glycerol are viable carriers for demineralized bone matrices. As both de-mineralized bone matrices tested provided an adequate osteoconductive matrix and showed some, although limited, osteoinductive capacity, these materials should be used in clinical practice only as bone graft extenders or enhancers.     

A radiographical and biomechanical study of demineralized bone matrix implanted into a bone defect of rat femurs with and without bone marrow

Repair of large bone defects represents a challenge to orthopedic surgery since autogenous graft is not available in large amounts. Demineralized bone matrix (DBM) which contains bone morphogenic protein, a potent osteoinductive glycoprotein, and collagen, an osteoconductive matrix, may be an effective substitute for these graft materials. Bone marrow which contains osteoprogenitor cells could potentiate the osteoinductive and osteoconductive properties of demineralized bone matrix. This study tested the ability of demineralized bone matrix with and without bone marrow to bridge large segmental defects, and evaluated the results both radiographically and biomechanically as compared to autogenous (isogeneic) cancellous bone graft. Demineralized bone-matrix segments implanted into a plated femoral segmental defect in rats resulted in firm union in most animals. Bone marrow significantly enhanced bone formation of demineralized bone-matrix implants at an early stage but with time, differences between bone marrow-augmented and bone marrow-deprived demineralized bone implants were no longer demonstrable radiographically and biomechanically. Newly formed bone had about 50% of the strength of the contralateral control bones. Femurs implanted with cancellous bone isografts had similar evidence of absolute union rate, radiographic and mechanical properties as DBM-implanted femurs.     

Induction of endochondral bone by demineralized bone matrix from diabetic rats

Summary In this investigation we examined the osteoinductive potential of demineralized bone matrix derived from chronically diabetic (streptozotocin-induced) rats. Long-Evans rats (28–31 days) were made diabetic with a single injection of streptozotocin (65 mg/kg) and provided food and waterad lib for 2 months. Diaphyseal shafts of femurs and tibias removed from the diabetic rats and their sibling controls were dehydrated, pulverized, sieved to 74–420 μm particles, and demineralized Matrix was then bioassayed for its ability to induce endochondral bone on day 11 following subcutaneous implantation over the thorax of Long-Evans rats. The resulting plaques of tissue were subjected to histological analysis, determination of alkaline phosphatase activity, and calcium content. Bone matrix derived from diabetic animals proved to be a significantly better inducer of endochondral bone than did control matrix.     

Neo-osseous flaps using demineralized allogeneic bone in a rat model

Surgical reconstruction with revascularized bone grafts can be compromised by donor tissue limitations and may be refined by prefabrication of compound neoflaps using bone substitutes. The principal suitability of demineralized allogeneic bone (DALB) slabs in fabricating neo-osseous flaps based on the inferior epigastric vascular system was studied and compared with neoflaps with autologous bone (AUB). In 45 rats, the histological pattern of bone formation in response to angiogenesis induced by vessel implantation was assessed, and characteristics of vascularization of the neoflap were studied microangiographically at 2, 4, 6, and 8 weeks. Histological techniques included decalcified and nondecalcified sections, as well as intravital polyfluorochrome labeling. Blood flow of the neoflap was also assessed quantitatively using 15-microm microspheres labeled with technetium 99-methylene diphosphate (99-MDP) 8 weeks after flap fabrication. Although the DALB neoflaps showed consistent bone formation and neovascularization, the bone regeneration process was delayed distinctly in comparison with AUB. Microangiographically, however, no differences between the two types of grafts became apparent during all time periods tested. Furthermore, the radioactivity of the DALB neoflap, which means bone blood flow per dry weight, was significantly higher than in AUB grafts and even more than that of intact iliac bone (p = 0.001). The exact meaning of elevated blood flow in DALB and similar degrees of vascularization corresponding to native AUB grafts remains to be determined, but may be a sign of ongoing bone formation resulting in a suitable DALB-containing neo-osseous flap in the long term. The authors findings support that allogeneic bone could be a potential substitute for AUB in creating a prefabricated neo-osseous flap.     

The efficacy of different commercially available demineralized bone matrix substances in an athymic rat model

OBJECTIVE: Bone graft substitutes have been developed because there is a limited supply of autogenous graft and the harvesting of iliac crest bone graft is associated with significant morbidity. Currently, there are a number of different commercially available demineralized bone matrix (DBM) products available that have been prepared by different methods and have different carriers. The objective of this study was to compare eight different commercially available DBM products. METHODS: Eight different DBMs were used to attempt a spinal fusion between the L4-L5 transverse processes in athymic rats. There were 10 rats in each group, and 5 rats were killed at both 4 and 8 weeks. Radiographic and histologic analyses were performed. Manual palpation testing was also performed. RESULTS: At 4 weeks, Osteofil Paste had the highest radiographic scores, whereas Grafton Putty had the best radiographic scores at 8 weeks. Conversely, the spines implanted with Allomatrix had the lowest radiographic scores at both 4 and 8 weeks. In regard to forming a spinal fusion confirmed by manual palpation, Osteofil Paste was the most effective at 4 weeks, whereas Grafton Flex and Grafton Putty had the highest rate of fusion at 8 weeks. Conversely, the lowest rates of fusion were seen in the Allomatrix and Grafton Crunch groups. Statistical analysis showed that there were significant differences among the groups seen on radiographs and by manual palpation. Qualitative differences could be appreciated between the groups histologically. CONCLUSION: Significant differences exist among commercially available DBMs in forming a spinal fusion in an athymic rat.     

掺锶硫酸钙及DBM复合材料骨修复能力评价

[目的]制备一种掺锶硫酸钙与DBM复合的骨修复材料,为临床提供一种成骨活性优良的骨修复材料。[方法]制备掺锶硫酸钙与DBM复合的活性骨修复材料,其中锶元素的量为钙含量的6%,掺锶硫酸钙与DBM按体积比1∶1复合。观察兔股骨髁骨缺损模型修复效果,利用X线片观察缺损区域的新骨形成情况,采用MicroCT评价局部的骨量和骨质变化,对组织切片予以Van Gieson(VG)复合染色法染色进行组织形态学观察,荧光双标观测计算成骨速率。[结果]术后动物无死亡,所有兔子切口无渗血或出现分泌物状况,术后活动正常。从MicroCT的BV/TV结果看出实验组在12周时缺损区域已经恢复;掺锶CaSO_4复合材料组缺损部位已经基本修复,CaSO_4组未完全修复。VG组织学观察显示各组均有新生骨小梁生成,荧光双标观测得到的矿化沉积率显示复合材料组新骨生长突出,矿化沉积率为(2.31±0.13)μm/d。[结论]通过MicroCT、VG组织学染色以及荧光双标观测对兔子股骨髁缺损修复效果进行评价,发现复合材料具有较好的骨缺损修复能力。     

Effect of glass bioactivity on new bone development induced by demineralized bone matrix in a rat extraskeletal site

The effects of two kinds of bioactive glass and two kinds of phosphate-free glass on new bone development induced by dernineralized bone matrix (DBM) were studied in the rat abdominal muscle pouch model. After 8 weeks' implantation histomorphometric analysis revealed that the amount of new bone in DBM combined with bioactive glass was comparable to DBM without bioactive glass. DBM grafts combined with phosphate-free glass showed significantly less new bone formation. Scanning electron microscopic examination confirmed that new bone bonded to the surface of bioactive glass. The release of ions from the glass seemed to slow down after new bone had bonded to it. Exclusion of phosphate from a bioactive glass resulted in loss of ability to develop the Ca,P-rich surface layer needed for bone bonding. contains BMP and other growth factors capable of inducing bone formation when implanted in various sites in laboratory animals [17–19].Bioactive glasses have several beneficial properties as a bone substitute. The crystal chemistry of the surface formed in in vivo apatite contributes to a high bone bonding rate [6, 8], and the rate of reactivity can be controlled by choice of glass composition [2]. In addition, bonding of glass to soft tissues has been reported [7, 20].We have previously reported formation of new bone directly on bioactive glass, induced by DBM in rat muscle tissue [13]. In the present study, the effects of four different glasses on new bone formation in DBM were studied in an extraskeletal site.     

The effect of demineralized bone matrix gel on bone ingrowth and fixation of porous implants

The presence of demineralized bone matrix (DBM) gel did not enhance or accelerate attachment strength or bone ingrowth and resulted in a significant decrease in implant interface attachment strength at 3 weeks. Hydroxyapatite (HA) coating resulted in significant increases in interface shear strength and bone ingrowth compared with non-HA-coated porous implants at all time periods. The HA-coated implants achieved greater attachment strength and bone ingrowth at earlier time periods and maintained greater attachment strength at long-term periods. The results of this study indicate that in the presence of a good bone-implant interference fit, there is no beneficial effect in applying DBM gel to a porous-coated or HA-coated porous implant surface. The small amount that can be applied and the degree of osteoinductivity of DBM seem to preclude it from having a significant biologic effect.     

骨蛋白强化脱钙骨基质板块修复犬长骨节段性骨缺损

目的 :研究骨蛋白 (boneprotein ,BP)强化脱钙骨基质 (demineralizedbonematrix ,DBM) (BP/DBM )板块在修复节段性骨缺损中的作用。方法 :在犬双侧桡骨中段各做一 1 5cm的骨膜骨缺损 ,分别植入板块状BP/DBM ,DBM ,自体髂骨块及留置空白 ,观察时间为 4个月。结果 :BP/DBM植入组有 3例完全骨愈合 (3 / 5 ) ,自体骨移植组只有 3例部分骨愈合 ,单纯DBM组及空白组未见骨愈合。生物力学测试 :术后 4个月BP/DBM组新生骨极限压缩强度值最高 ,已达到正常桡骨组织的 48%。BP/DBM组新生骨为成熟的板状骨。结论 :BP/DBM板块可促进节段性骨缺损的修复。     

Osteoinductivity of commercially available demineralized bone matrix. Preparations in a spine fusion model

BACKGROUND: Although autogenous bone is the most widely used graft material for spinal fusion, demineralized bone matrix preparations are available as alternatives or supplements to autograft. They are prepared by acid extraction of most of the mineralized component, with retention of the collagen and noncollagenous proteins, including growth factors. Differences in allograft processing methods among suppliers might yield products with different osteoinductive activities. The purpose of this study was to compare the efficacy of three different commercially available demineralized bone matrix products for inducing spinal fusion in an athymic rat model. METHODS: Sixty male athymic rats underwent spinal fusion and were divided into three groups of eighteen animals each. Group I received Grafton Putty; Group II, DBX Putty; and Group III, AlloMatrix Injectable Putty. A control group of six animals (Group IV) underwent decortication alone. Six animals from each of the three experimental groups were killed at each of three intervals (two, four, and eight weeks), and the six animals from the control group were killed at eight weeks. At each of the time-points, radiographic and histologic analysis and manual testing of the explanted spines were performed. RESULTS: The spines in Group I demonstrated higher rates of radiographically evident fusion at eight weeks than did the spines in Group III or Group IV (p < 0.05). Manual testing of the spines at four weeks revealed variable fusion rates (five of six in Group I, two of six in Group II, and none of six in Group III). At eight weeks, all six spines in Group I, three of the six in Group II, and no spine in Group III or IV had fused. Histologic analysis of the spines in Groups I, II, and III demonstrated varying amounts of residual demineralized bone matrix and new bone formation. Group-I spines demonstrated the most new bone formation. CONCLUSIONS: This study demonstrated differences in the osteoinductive potentials of commercially available demineralized bone matrices in this animal model.     

Evaluation of osteogenic cell differentiation in response to bone morphogenetic protein or demineralized bone matrix in a critical sized defect model using GFP reporter mice

We evaluated the osteoprogenitor response to rhBMP‐2 and DBM in a transgenic mouse critical sized defect. The mice expressed Col3.6GFPtopaz (a pre‐osteoblastic marker), Col2.3GFPemerald (an osteoblastic marker) and α‐smooth muscle actin (α‐SMA‐Cherry, a pericyte/myofibroblast marker). We assessed defect healing at various time points using radiographs, frozen, and conventional histologic analyses. GFP signal in regions of interest corresponding to the areas of new bone formation was quantified using a novel computer assisted algorithm. All defects treated with rhBMP‐2 healed. In contrast, the majority of the defects in the DBM (27/30) and control (28/30) groups did not heal. Quantitation of pre‐osteoblasts demonstrated a maximal response (% GFP+ cells/TV) in the Col3.6GFPtopaz mice at day 7 (7.2% ± 6.0, p < 0.05 compared to days 14, 21, 28, and 56). The maximal response of the Col2.3GFP cells was seen at days 14 (8.04% ± 5.0) and 21 (8.31% ± 4.32), p < 0.05. In contrast, DBM and control groups showed a limited osteogenic response at all time points. In conclusion, we demonstrated that the BMP and DBM induce vastly different osteogenic responses which should influence their clinical application as bone graft substitutes. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1120–1128, 2014.     

Calcificationin vitro of demineralized bone matrix

Collagen was prepared from compact sheep bone by decalcification with EDTA and from rat tail tendons by acetic acid extraction and reconstitution with NaCl. The deposition of apatite in sheep bone collagen in a metastable calcification solution was studied chemically and by electron microscopy. The bone collagen was shown to be a good nucleation catalyst for mineral deposition, while rat tail collagen was a poor catalyst. Mineral deposition in bone collagen occured in two separate kinetic phases, a rapid phase of nucleation and crystal growth, giving rise to small calcified islands, and a second slow phase, ascribed to growth in regions not involving the catalytic sites. This second phase of mineral deposition is considered to be the result of impaired ion diffusion through the closely-aligned collagen fibrils, thus leaving large areas of the collagen free of mineral even though the buffer remains highly supersaturated. Electron micrographs suggested that the catalytic sites might be in some relationship to the 640 Å periodicity of collagen, but a role for non-collagenous material bound to the collagen has not been excluded.The poor catalytic activity of reconstituted collagen was not due to the presence of loosely-bound inhibitors, although inhibitors could be strongly bound to this type of collagen and be absent from bone collagen. The differences in catalytic activity may have a bearing on physiological calcification. A more general hypothesis for nucleation of a mineral phase in biological systems is required.This work was supported in part by the European Atomic Energy Community (EURA-TOM), Brussels, Belgium.     

Osteoinductive activity of demineralized bone matrix and deprotenized bone derived from human avascular necrotic femoral head

Objective: To observe the osteoinductive activity of demineralized bone matrix (DBM) and deprotenized bone (DPB) made from human avascular necrotic femoral head. Methods: The femoral head was cut into pieces with the size of 3 mm×3mm×5 mm, which were made into DBM and DPB. These two kinds of biomaterials were cocultured with human bone mesenchymal stem cells (hBMSCs). Monolayer cells without biomaterials were cultured as control. Proliferative activity of hBMSCs was evaluated on days 1, 3, 5, 7 and 14. The concentration of alkaline phosphatase (ALP), osteocalcin (OC), and Ca2+ were detected on days 1, 7, 14 and 21. Results: Cells cultured in DBM showed higher proliferative activity than did in DPB and monolayer cells (F= 39.773, P<0.01). DBM and DPB also had osteoinductive activity. The concentrations of ALP (F=93.162, P<0.01), OC (F=236.852, P<0.01),Ca2+(F=80.711, P<0.01) of DBM group were significantly higher than that of DPB and control groups. Conclusions: In vitro, DBM and DPB made from avascular necrotic femoral head have osteoinductive activity when cocultured with hBMSCs, and the former is stronger than the latter.     

Use of demineralized bone matrix in children

Eighty five clinical observations of the authors have shown autotransplants demineralized in the solution of hydrochloric acid to possess pronounced osteoinductive properties. Their ability to induce osteogenesis was mostly pronounced in simultaneous plasty with a demineralized and frozen bone. Reconstructive processes are perfectly completed within 18-24 months after operation.     

Efficacy comparison of Accell Evo3 and Grafton demineralized bone matrix putties against autologous bone in a rat posterolateral spine fusion model

Background Context

Spinal fusion procedures are intended to stabilize the spinal column for a multitude of disorders including abnormal curvature, traumatic instability, degenerative instability, and damage from infections or tumors. As an aid in the bone healing response, bone graft materials are used to bridge joints for arthrodesis and promote unions in pseudoarthrosis. Currently, the gold standard for stabilizing fusion masses in spinal procedures involves using the osteogenic, osteoinductive, and osteoconductive properties of autologous iliac crest corticocancellous bone. However, considerable morbidity is associated with harvesting the autologous graft. Donor site complications including infection, large hematomas, and pain have been reported at rates as high as 50% (Boden and Jeffrey, 1995). Biologically, the rate of bone repair dictates the rate at which the fusion mass will unite under autologous graft conditions.

Serum albumin enhances bone healing in a nonunion femoral defect model in rats: a computer tomography micromorphometry study

Purpose

Blood-derived proliferative factors such as platelet rich plasma or activated plasma are promising adjuvants for bone grafts. Our earlier studies showed that serum albumin itself can markedly enhance the proliferation of stem cells on bone allograft and postulated that albumin coating alone may improve bone graft integration in vivo.

Methods

Two femoral defect models were performed in adult male Wistar rats. In the critical size model a six millimetre gap was created in the midshaft of the femur and fixed with plate and screws, while a nonunion model was established by the interposition of a spacer in the osteotomy for four weeks which resulted in compromised healing and nonunion. Albumin coated and uncoated grafts were placed into the defects. Bone healing and morphometry were evaluated by μCT and histology four weeks after implantation of the grafts.

Results

In the critical size model none of the bone grafts were able to bridge the defect, and graft resorption was the typical outcome. In the nonunion model regular uncoated grafts had a low union rate (two out of six), which increased markedly when albumin coating was applied (six out of eight). Trabecular thickness and pattern factor improved significantly in the albumin coated group versus uncoated or empty controls.

Conclusions

Our results showed that serum albumin coating of bone grafts can enhance the remodelling and efficacy of treatment in a nonunion model.     

Restoration of bone defect and enhancement of bone ingrowth using partially demineralized bone matrix and marrow stromal cells

PURPOSE: This study aimed to investigate the capability of combining marrow stromal cells (MSC) and partially demineralized bone matrix (PDBM) to fill bone defect and enhance bone ingrowth using a canine non-weight-bearing gap model. METHODS: Custom-made implants with 3mm gap between the porous surface and the host bone were used. The implants were inserted into the distal femurs of 25 mongrel dogs and the gaps were randomly assigned to be filled with culture-expanded autologous MSC-loaded PDBM, autograft, fresh-frozen allograft, PDBM alone, or nothing as controls. Histomorphometry using backscattered scanning electron microscopic examination, and mechanical push-out test were performed at 6 months after surgery. RESULTS: Histomorphometry showed that amounts of bone regeneration in the gap and bone ingrowth into the porous-coated surface in the MSC-loaded PDBM-treated group were comparable to those of autograft-treated group and were significantly greater than those of allograft-treated, PDBM-treated, or non-grafted groups. Mechanical test showed the same differences. CONCLUSION: The results of this study showed that combining PDBM and autologous culture-expanded MSC restored bone stock and enhanced bone ingrowth into the porous-coated area in a canine non-weight-bearing gap model. This combination may provide an option for reconstructing bone defect when we perform a cementless revision arthroplasty.     

Posterolateral lumbar spine fusion using a novel demineralized bone matrix: a controlled case pilot study

Introduction

Intertransverse posterolateral fusion along with instrumentation is a common technique used for spinal fusion. Iliac crest bone graft (ICBG) offers good fusion success rates with a low risk for disease transmission but is, however, linked with certain morbidity. In an effort to eliminate or reduce the amount of iliac graft needed, bone substitutes including demineralized bone matrix (DBM) have been developed. This study evaluates a novel DBM (Accell Connexus^®) used in one or two-level instrumented posterolateral lumbar fusion.

Materials and methods

A total of 59 consecutive patients were studied as two groups. Group 1 consisted of 33 patients having Accell Connexus^® used to augment either ICBG or local decompression material. Group 2 consisted of 26 consecutive patients, operated prior to the introduction of this novel DBM, having either ICBG alone or local decompression material. Fusion was assessed by two independent observers, blinded to graft material, using standardized criteria found in the literature. All adverse events were recorded prospectively.

Results

The results show no statistically significant differences between the two groups in fusion rates, complications, surgery duration, ODI, or pain on VAS. Logistical regression showed no relation between fusion and age, smoking status or comorbidities. Furthermore, no adverse events related to the use of the novel DBM were observed.

Conclusion

The results from this study demonstrate that the novel DBM presented performs equally as well as that of autologous bone, be it either ICBG or a local decompression material, and can therefore be used as a graft extender.

     

Use of demineralized bone matrix in the extremities

Autologous bone graft is considered as the gold standard for all indications for bone grafting procedures but the limited availability and complications in donor site resulted in seeking other options like allografts and bone graft substitutes. Demineralized bone matrix (DBM) is an allograft product with no quantity limitation. It is an osteoconductive material with osteoinductive capabilities, which vary among different products, depending on donor characteristics and differences in processing of the bone. The purpose of the present review is to provide a critical review of the existing literature concerning the use of DBM products in various procedures in the extremities. Clinical studies describing the use of DBM alone or in combination with other grafting material are available for only a few commercial products. The Level of Evidence of these studies and the resulting Grades of Recommendation are very low. In conclusion, further clinical studies of higher quality are required in order to improve the Recommendation Grades for or against the use of DBM products in bone grafting procedures.     

Evaluation of a new formulation of demineralized bone matrix putty in a rabbit posterolateral spinal fusion model

Background contextAlternatives to autologous bone graft (ABG) with osteoconductive, osteoinductive, and osteogenic potential continue to prove elusive. Demineralized bone matrix (DBM) however, with its osteoconductive and osteoinductive potential remains a viable option to ABG in posterolateral spine fusion.PurposeTo compare the efficacy of a new formulation of DBM putty with that of ABG in a rabbit posterolateral spinal fusion model.Study designEfficacy of a new formulation of DBM was studied in an experimental animal posterolateral spinal fusion model.MethodsTwenty-four male New Zealand White rabbits underwent bilateral posterolateral spine arthrodesis of the L5–L6 intertransverse processes, using either ABG (control group, n=12) or DBM (DBM made from rabbit bone) putty (test group, n=12). The animals were killed 12 weeks after surgery and the lumbar spines were excised. Fusion success was evaluated by manual palpation, high resolution X-rays, microcomputed tomography imaging, biomechanical four-point bending tests, and histology.ResultsTwo animals were lost because of anesthetic related issues. Manual palpation to assess fusion success in the explanted lumbar spines showed no statistical significant difference in successful fusion in 81.8% (9/11) of DBM group and 72.7% (8/11) of ABG group (p=.99). Reliability of these assessments was measured between three independent observers and found near perfect agreement (intraclass correlation cofficient: 0.92 and 0.94, respectively). Fusion using high resolution X-rays was solid in 10 of the DBM group and 9 of the ABG group (p=.59). Biomechanical testing showed no significant difference in stiffness between the control and test groups on flexion, extension, and left lateral and right lateral bends, with p values accounting for .79, .42, .75, and .52, respectively. The bone volume/total volume was greater than 85% in the DBM treated fusion masses. Histologic evaluation revealed endochondral ossification in both groups, but the fusion masses were more mature in the DBM group.ConclusionsThe DBM putty achieved comparable fusion rates to ABG in the rabbit posterolateral spinal fusion model.