Effect of sterilization on osteoinduction Comparison of five methods in demineralized rat bone

The aim of this study was to find a safe, effective sterilization method that does not destroy the bone-inductive capacity of demineralized bone implants. Five sterilizing agents were tested in rats. Implants procured and processed under sterile conditions served as controls. New bone formation was evaluated by determining dry weight, calcium content, and Sr-85 incorporation of the induced ossicles.

Glutaraldehyde solution, formaldehyde gas, and ethylene oxide destroyed almost all the bone-inductive capacity. Irradiation by 2.5 Mrads Co-60 resulted in a loss of about half of the inductive capacity. Merthiolate (0.18 per cent) was the only sterilizing agent that did not reduce the bone-inductive capacity of the demineralized implants. Because merthiolate is not sporicidal, gamma irradiation appears to be the most appropriate sterilizing agent for demineralized bone in clinical use.     

Effect of sterilization on osteoinduction. Comparison of five methods in demineralized rat bone

The aim of this study was to find a safe, effective sterilization method that does not destroy the bone-inductive capacity of demineralized bone implants. Five sterilizing agents were tested in rats. Implants procured and processed under sterile conditions served as controls. New bone formation was evaluated by determining dry weight, calcium content, and Sr-85 incorporation of the induced ossicles. Glutaraldehyde solution, formaldehyde gas, and ethylene oxide destroyed almost all the bone-inductive capacity. Irradiation by 2.5 Mrads Co-60 resulted in a loss of about half of the inductive capacity. Merthiolate (0.18 per cent) was the only sterilizing agent that did not reduce the bone-inductive capacity of the demineralized implants. Because merthiolate is not sporicidal, gamma irradiation appears to be the most appropriate sterilizing agent for demineralized bone in clinical use.     

Effects of gamma irradiation on osteoinduction associated with demineralized bone matrix

Gamma irradiation is frequently used to sterilize implanted devices but has limitations when used on biologically active materials and composites. In this study, we have evaluated the changes of biological activity of demineralized bone matrix (DBM) in the dry state and in the presence of aqueous and non‐aqueous carriers while exposed to various levels of ionizing radiation. The activity of DBM in the dry state remains relatively stable with only a small loss of activity. Composites of DBM with a carrier such as lecithin, to which no water has been added, lose activity at approximately the same rate as DBM in the anhydrous form. In composites that contain water, the loss of activity occurs even at much lower levels of radiation exposure. Gamma irradiation does not change cell attachment to the DBM matrix but has an influence on both stem cell and osteoprecursor cell proliferation rates. Because of the limitations imposed by radiation, it seems most practical to handle DBM aseptically throughout the procedures of compositing pastes, putties, or suspensions, and only if necessary exposing the inert excipients to radiation sterilization prior to mixing. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:75–82, 2008     

微波加热对脱钙骨基质诱导活性的影响

为了观察不同时间、温度微波加热后脱钙骨基质诱导活性的变化,作者以２４只成年纯种新西兰白兔为实验对象,随机分为２周（７只）、４周（７只）８周（５只）、１２周（５只）四个时间组。取兔左下肢胫骨用电锯锯成长度为０．８ｃｍ的移植骨６块,行微波加热。按不同时间、温度分为：３７℃、３０分钟组为１组;４５℃、３０分钟为Ⅱ且：６０℃、３０分钟为Ⅲ组;７５℃、３０分钟为Ⅳ组;７５℃、６０分钟为Ⅴ组;１００℃、３０分     

Effect of Er: YAG laser holes on osteoinduction in demineralized rat calvarial allografts

Massive cortical autografts and allografts have been found to incorporate into host bone very slowly and thus are subject to complications such as fatigue fracture and infection. In order to understand and improve the process of osteogenesis in these types of bone grafts, a new experimental model was developed using bone discs from rat calvaria prepared by demineralization and drilling of 0.5 mm diameter holes with a pulsed, 2.94 μm wavelength Erbium: Yttrium-Aluminum-Garnet laser. Four types of bone discs were analyzed: untreated (Type I), demineralized (Type II), laser-ablated (Type III), and laser-ablated then demineralized (Type IV). The discs were transplanted into a subcutaneous site in adult Sprague-Dawley rats and followed for as long as 6 weeks. Histologic analysis of the discs at weekly intervals with use of hematoxylin and eosin staining confirmed the presence of new bone growth in Type-II and Type-IV discs. The amount of new bone growth in each disc was estimated by determining the mineral x-ray attenuation coefficient, which is proportional to mineral density, from digitized radiographs of the discs. The results showed that the processes of demineralization (p < 0.001) and laser ablation with demineralization (p < 0.05) were both significant in enhancing new bone growth in this model. This study demonstrated that osteoinduction can be fostered in cortical bone through the processes of demineralization and laser ablation. To the extent that laser ablation may allow maintenance of structural integrity while altering the surface geometry in such a way as to promote ingrowth of new bone, this experimental model represents an advance in understanding how osteogenesis in cortical bone grafts might be improved.     

Xenogenic demineralized bone matrix: osteoinduction and influence of associated skeletal defects in heterotopic bone formation in rats

Demineralized bone matrix (DBM) was ectopically implanted in 36 male Wistar rats. In 18 of the animals a bone defect in the femoral condyles was also created: the left was filled with DBM and the right was left empty as a control. The animals were killed after 2, 4 and 6 weeks and new bone was histologically evaluated, comparing ectopic bone formation with or without distant bone injury. Results showed: (1) osteoinductivity of xenogenic DBM, and (2) earlier mineralization of ectopically implanted DBM in the group with associated skeletal injury. Our results show that xenogenic bone matrix acts as an osteoinductive material and that skeletal injury improves osteogenesis at distant sites. Résumé  Sur 36 rats malesWistar, la matrice osseuse déminéralisée (DBM était ectopiquement implantée. Dans le même temps un défaut osseux au niveau des condyles fémoraux était réalisé sur 18 d’entre eux: à gauche ils étaient remplis avec la DBM et à droite, ils étaient laissées vides. Les rats furent sacrifiés après 2, 4 et 6 semaines et le nouveau tissu osseux était évalué histologiquement en comparant la formation du tissu osseux ectopique avec ou sans la lésion de l’os. Les résultats démontrent : (1) l’ostéoinductivité de la DBM xénogénique, (2) la minéralisation plus rapide de la DBM implantée ectopiquement dans le groupe avec lésion du squelette. Nos résultats démontrent que la matrice osseuse xénogénique agit comme un materiel ostéoinductif et que les lesions du squelette améliorent l’ostéogenèse des sites distants. Accepted: 19 September 1998     

Age effects on bone induction by demineralized bone powder

It has been previously shown that osteoinduction by demineralized bone powder (DBP) in the rat decreases as the age of the recipient animal increases. In the present study, the effects of age on osteoinduction by DBP were evaluated in the rat by varying the age of the donor and the recipient animal. Cartilage and bone formation in subcutaneous pouches was assessed using a histologic grading technique in which a composite score was derived from analysis of multiple histologic sections from each specimen. The results confirm the previously reported decrease in osteoinduction in middle-aged adult animals compared with younger ones. However, DBP prepared from middle-aged adult rats was more inductive than that prepared from either prepubertal or young postpubertal animals. The latter result contradicts the widely held belief that demineralized bone matrix from younger animals is more inductive than that from older ones. This finding may help to further elucidate the mechanism of ectopic bone formation and lead to more inductive bone graft substitutes for human use.     

Effect of demineralized bone matrix on polymorphonuclear leukocyte degranulation.

The potential use of allogenic demineralized bone matrix to augment or treat bone defects or nonunions in animals and humans is currently being investigated. Demineralized bone matrix induces osteogenesis by a multistep cascade of endochondral ossification that is mediated by bone-induction factors. The migration and activation of polymorphonuclear leukocytes appear to be critical in the initiation of the cascade of osteogenesis induced by demineralized bone matrix. This study examined the effects of demineralized bone matrix on the degranulation of polymorphonuclear leukocytes. Demineralized bone matrix stimulated the release of polymorphonuclear leukocyte-specific, but not azurophilic, granules in a time and dose-dependent manner. The ability of the bone matrix to induce this degranulation was independent of its size and species. The mechanism by which this degranulation occurs is not completely understood; however, it is known that it does not occur by means of a receptor that requires guanidine triphosphate-dependent regulatory proteins as does polymorphonuclear-leukocyte degranulation induced by N-formyl peptide. The factor that stimulates degranulation is not type-I collagen but rather appears to be a cytokine that has a heparin-binding domain and a molar mass of 10-70 kDa. Loss of the ability of demineralized bone matrix to induce degranulation of polymorphonuclear leukocytes correlated positively with the loss of its ability to induce bone formation.     

低浓度过氧化氢预处理骨髓间充质干细胞对创面愈合的促进作用

目的:观察低浓度过氧化氢(H2O2)预处理骨髓间充质干细胞(BMSCs)移植到大鼠组织缺损创面后BMSCs在创面的存活情况及其对创面的促愈合作用.方法:75只SD大鼠,于背部中线靠颈侧制备直径1 cm的圆形全层皮肤缺损.按随机数字表法分为3组(每组25只):生理盐水组、单纯BMSCs组和H2O2预处理BMSCs组.在伤后即刻,分别经尾静脉注射等体积(0.5 ml)的生理盐水、CM-Dil标记的BMSCs悬液或50 u mol/L H2O2预处理12 h并经CM-Dil标记的BMSCs.伤后0～15d创面拍照,用Image Proplus 5.0图像分析软件分析图像,计算创面愈合率;伤后1、2、5d荧光显微镜下观察两BMSCs组创面BMSCs的数量;CD31免疫组化染色观察创面微血管密度的变化.结果:①创面干细胞数量:移植H2O2预处理的BMSCs后,创面干细胞数量明显多于单纯BMSCs移植组(P＜0.05和P＜0.01).与伤后第1天比较,单纯BMSCs组伤后第2天和第5天创面干细胞数量显著减少(P＜0.05);而预处理组创面干细胞数量虽然也呈进行性减少,但3个时相点创面的干细胞数量差异没有显著性(P＞0.05).②创面愈合率:创伤后5～15d,单纯BMSCs组和H2O2预处理BMSCs组创面愈合率均明显高于生理盐水组(P＜0.05或P＜0.01);创伤后5～10d,H2O2预处理组创面愈合率明显高于单纯BMSCs组(P＜0.05).⑧创面微血管数量:CD31免疫组化结果显示,伤后3d,两BMSCs治疗组之间血管数量差异无显著性;伤后5、7、10 d,H2O2预处理组创面微血管数量显著多于单纯BMSCs组(P＜0.05).结论:BMSCs经低浓度H2O2预处理再移植到创面,可以延长移植干细胞在创面的存活时间,并具有更好的促进创面愈合和血管生成的修复潜能.     

冷冻、冻干、辐照对骨诱导活性的影响

[目的]研究冻干、冷冻、辐照对骨诱导活性的影响。[方法]收取胫骨上段松质骨,加工成大小约0.5 cm×0.5 cm×0.5 cm的骨块,随机分为五组:新鲜骨组、冷冻组、冻干组、冻干+辐照组以及冷冻+辐照组,用免疫组化法测定骨细胞因子BMP、bFGF、β-TGF含量的变化。[结果]新鲜骨及冷冻骨、冻干骨均有细胞因子表达,但冻干骨表达的程度着色较深,表达阳性的细胞位于骨小梁的表面。辐照后,BMP、bFGF、β-TGF表达减少。[结论]辐照可减少骨的诱导活性,但仍保留部分骨诱导成分。     

Induced healing of aneurysmal bone cysts by demineralized bone particles

Two cases of induced healing of aneurysmal bone cyst (ABC) following intralesional implantation of a bone paste made of autogeneic bone marrow and allogeneic bone powder are reported. The calcaneum in one case and the superior pubic ramus in the other were blown out by an ABC and would have required extensive surgery. Via a minimal exposure, the cyst was partially evacuated and filled with an admixture of a partially demineralized bone particles with bone marrow. Ossification of the peripheral shell was the first sign of healing and was observed within the first 3 postoperative months. Successful healing was observed in both cases. The rationale underlying this intralesional treatment was that the bone grafting material might reverse ABC expansion by promoting ossification through a bone induction mechanism. The concept of this treatment was to retain the ABC tissue, using its own intrinsic osteogenic potential to promote healing. By triggering intralesional new bone formation, the bone paste represented an effective means to reverse the expanding phase of ABC. The particulated bone allograft was easy to handle and to introduced in an irregular cavity. Moreover, as a complete cyst evacuation was not required, a minimal surgical approach could be used so that the risks and morbidity associated with an extensive approach were reduced. Its use is of particular interest in poorly accessible areas like the pelvis and spine.     

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.     

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.     

Cellular events associated with the induction of bone by demineralized bone

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

Treatment of aneurysmal bone cysts by introduction of demineralized bone and autogenous bone marrow

BACKGROUND: On the assumption that an aneurysmal bone cyst has an intrinsic potential to heal by ossification, a new, minimally invasive protocol was developed. Demineralized bone powder mixed with bone-marrow aspirate was introduced into the cyst to halt the expansion phase and to allow the cyst to ossify. We hypothesized that, in order to induce bone-healing, cells from the cyst are needed to respond to the inductive material but that curettage or extensive surgery is not necessary. The goals of the present study were to assess cyst-healing and to determine the prevalence of recurrence associated with this new procedure. METHODS: Thirteen biopsy-proven primary aneurysmal bone cysts were entered through a small incision, and a paste of demineralized bone and autologous bone marrow was introduced with an applicator. The study group included three male and ten female patients with a mean age of 16.6 years. The cyst was located in a long bone in six patients, the pelvis in five patients, and the scapular glenoid and the calcaneus in one patient each. Five patients had not received treatment previously, whereas one had had a preoperative embolization and seven had recurrent lesions that had been treated previously. RESULTS: After a mean duration of follow-up of 3.9 years, healing was achieved in eleven patients. CONCLUSIONS: This minimally invasive method is able to promote the self-healing of a primary aneurysmal bone cyst. As no curettage is required, the proposed treatment avoids extensive surgery and blood loss and is convenient for the treatment of poorly accessible lesions such as those occurring in the pelvis. LEVEL OF EVIDENCE: Therapeutic Level IV.     

Enhancement of bone bonding to bioactive ceramics by demineralized bone powder.

In an attempt to enhance the bonding of bone to bioactive ceramics, allogeneic demineralized bone powder (DBP) was used in combination with bioactive ceramic implants in rabbit tibiae. Rectangular plates (10 x 15 x 2 mm) made of apatite-wollastonite-containing glass ceramics were implanted in the proximal metaphyses of the bilateral tibiae of 20 rabbits, with DBP packed into the medullary cavity. In the control group, only the plates of A-W GC were implanted in the bilateral tibiae of 20 rabbits. Four rabbits from each group were killed at two, four, eight, 12, and 25 weeks after implantation for the tensile test. Results of the tensile test and histologic examination of the undecalcified specimens by Giemsa surface stain and contact microradiography confirmed that DBP significantly accelerated the process of bone bonding to the implant and increased the strength of bone-implant bonding.     

一种高效制备工艺对脱钙骨基质性能的影响

目的 目前脱钙骨基质生产工艺耗时长效率低,浪费资源和成本.本研究在保存骨诱导活性的前提下,显著缩短了脱钙骨基质的工艺时间.方法 采用动态脱钙和二次换酸工艺制备脱钙骨基质,对pH值、钙含量进行评价.用裸鼠体内植入实验评价这种脱钙骨基质骨诱导活性.结果 材料的pH值为6.2±0.3,呈弱酸性.钙含量为(0.6±0.2)％,符合＜6％的标准.裸鼠体内植入后创面一期愈合,无不良反应发生.其骨诱导活性阳性,4周时可见大量的新生骨组织、骨髓样组织和软骨化成骨现象.结论 该工艺可以提高脱钙骨基质生产效率,不影响材料活性.