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 hydrogen peroxide on osteoinduction by demineralized bone

The osteoinductive capacity of demineralized bone matrix (DBM) has led to wide use of this material for surgical reconstruction. Preparation of DBM often includes sterilization with ethylene oxide, disinfection with various chemical agents, or irradiation. Exposure to hydrogen peroxide (H2O2) is used for both sterilization and bleaching of bone, the latter primarily for cosmetic reasons. We investigated the effect of H2O2, on the osteoinductive capacity of DBM. Cortical bone implants prepared from rat femurs were placed into 3% H2O2 solution. Control specimens were not exposed to H2O2. Bones were then lipid-extracted, demineralized, sterilized with ethylene oxide, and freeze-dried in an identical manner. Allografts were implanted into rat hosts for 1 to 3 weeks. Osteoinduction proceeded rapidly in implants not exposed to H2O2, with chondrocytes and new bone appearing in the implant. After 3 weeks, perforations in the implant were largely replaced with new bone. In contrast, osteoinduction did not occur in implants treated with H2O2. Perforations in H2O2-treated implants were filled with vascularized fibrous tissue, but no cartilage or bone. These findings reveal that H2O2 used for disinfection or bleaching of DBM can abolish its osteoinductive capacity in rats.     

Effect of gas-plasma sterilization on the osteoinductive capacity of demineralized bone matrix.

The current study evaluated the effect of low-temperature hydrogen peroxide gas plasma sterilization on the osteoinductive capability of human demineralized bone matrix using a rat model. Twelve athymic rats received three separate implants consisting of steam-sterilized demineralized bone matrix (negative control), sterile-harvest demineralized bone matrix (positive control), and gas-plasma-sterilized demineralized bone matrix. A demineralized bone matrix pellet from each sterilization group was placed individually into one of three separate soft tissue pockets created in the epaxial musculature of each rat. All 12 rats were euthanized 9 weeks after implantation. Each implantation site was removed along with 0.5-cm normal tissue around the implant. Histologic examination was done on each implant site to determine the presence or absence of new bone, cartilage, or bone marrow elements. All 12 sterile harvest demineralized bone matrix sites histologically contained new bone elements, whereas none of the negative control or gas plasma sterilized demineralized bone matrix sites contained any of these same elements. The results of this study indicate that demineralized bone matrix sterilized with low-temperature, gas-plasma sterilization loses its osteoinductive capacity in a manner similar to that of steam-sterilized demineralized bone matrix, making low-temperature, gas- plasma sterilization unsuitable as a method of secondary sterilization of demineralized bone matrix.     

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.     

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     

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     

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.     

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 various sterilization procedures on the osteoinductive properties of demineralized bone matrix]

To minimize potential infection following the transplantation of allogeneic bone, extremely rigorous selection of donors and careful processing and storage of samples are required. Other major problems related to allogeneic transplants, such as reduced osteogenic properties and immunological reactions, led to the development of demineralized bone matrix (DBM). This osteoinductive bone extract is largely free of antigens and is easy to produce. However, to eliminate the potential risk of infection, DBM should be sterilized prior to implantation. The purpose of this study was to investigate the influence of different sterilization techniques on the osteoinductive properties of DBM. A series of 76 cortical defects (drill holes) 0.6 cm in diameter in the tibiae of 11 Merino sheep were filled with DBM in addition to autogeneic and allogeneic cancellous bone. Prior to implantation DBM was sterilized by autoclaving, gamma irradiation, or application of ethylene oxide or ethyl alcohol. A further 12 drill holes were left empty as controls. The formation of new bone was examined 3 and 6 weeks postoperatively, using histological, fluorescent-optical and microradiographical techniques. The amount of newly formed bone was also quantified. Apart from autoclaved DBM all matrix grafts showed excellent new bone formation following sterilization, by far exceeding the formation with allogeneic cancellous bone.     

Effect of sterilization on bone morphogenetic protein

Demineralized bone matrix and bone morphogenetic protein have been used clinically to accelerate bone regeneration. However, the best method of sterilization has been the subject of controversy. Some investigators have used ethylene oxide, but others have reported that doses adequate for sterilization destroyed the osteoinductivity of demineralized bone matrix and that gamma irradiation was less harmful in this respect. We used partially purified bone morphogenetic protein and type-I collagen to investigate the effects of sterilization by ethylene oxide and gamma irradiation on the activity of bone morphogenetic protein. Osteoinductivity was reduced considerably after sterilization by gamma irradiation at 2.5 Mrad and by ethylene oxide at 37°C for 4 hours and at 55°C for 1 hour; however, the reduction induced by ethylene oxide at 29°C for 5 hours was about half of the control values. This study showed that ethylene oxide at 29°C for 5 hours can be used clinically for sterilization of bone morphogenetic protein. We also investigated the effect of gamma irradiation on bone morphogenetic protein and the collagen carrier separately and found that collagen was far more labile than bone morphogenetic protein.     

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.     

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。     

Immunosuppression with FK506 increases bone induction in demineralized isogeneic and xenogeneic bone matrix in the rat.

The aim of the present study was to investigate a systemic induction of bone formation in rats by immunosuppression with FK506 (1 mg/kg body weight intraperitoneally [ip]) in a model of osteoinduction of isogeneic and xenogeneic demineralized bone matrix (DBM) for a period of 28 days. In particular, alterations of in vitro cytokine synthesis and changes of lymphocyte subsets were studied. DBM was implanted intramuscularly in the abdominal wall of Lewis rats (seven per group). Blood was sampled on days -7, 0, 7, and 28 for determination of in vitro tumor necrosis factor a (TNF-alpha) synthesis and lymphocyte subsets by flow cytometry (CD3+, CD4+, CD8+, CD45+, ED9+, and Ia+ antibodies). Ossicles of de novo formed bone and the tibias were removed on day 28 after double tetracycline labeling for histomorphometric analysis. Immunosuppression with FK506 significantly decreased lipopolysaccharide (LPS)-stimulated in vitro cytokine synthesis after 7 days and 28 days (p < 0.05). Compared with control animals FK506 treatment significantly increased the volume of induced bone in isogeneic (2.1 +/- 0.3 mm3 vs. 10.8 +/- 0.9 mm3) and xenogeneic (O mm3 vs. 4.7 +/- 0.8 mm3) DBM. Bone histomorphometry of the tibias revealed that immunosuppression increased both bone formation and bone resorption, accompanied by a significant reduction in the relative trabecular area (Tb.Ar). FK506 caused a decrease in the counts of CD8+ T cells probably because of destruction or dislocation of these cells. This suggests that the amount of CD8+ cells and the degree of T cell activation in terms of mean fluorescence intensity (MFI) may be associated with bone metabolism. In support of this, statistical analysis revealed a significant positive correlation between parameters of bone formation as well as bone resorption and the CD4+/CD8+ ratio. There was a significant negative correlation between parameters of remodeling of the metaphysis of the tibia and induced bone volume (BV), respectively, and MFI values of CD3+/Ia+ cells. These findings suggest an important role of T lymphocytes in bone formation and bone resorption in vivo. FK506 caused a marked increase of bone formation in DBM. However, the conclusion that immunosuppression increases fracture healing warrants further investigation.     

异体骨垫与聚醚醚酮融合器在颈椎融合术中的对比研究

[目的]通过与聚醚醚酮(polyether ether ketone PEEK)颈椎融合器比较,评价同种异体骨垫在颈椎前路椎间融合术中的疗效。[方法]对46例颈椎病患者行颈椎前路减压、椎间融合内固定术,根据材料不同,分为同种异体骨垫组(A组)和聚醚醚酮颈椎融合器组(B组),术后随访24个月。参照日本骨科协会(JOA)评分标准,评价指标为颈椎间隙融合率,融合时间,手术时间,术中出血及排斥反应。[结果]术后24个月异体骨垫组JOA评分13.1分,高于聚醚醚酮融合器组的12.8分,但无统计学差异(P>0.05)。A、B组术后有效融合率分别为96.0%和95.2%,无统计学差异(P>0.05)。A组与B组比较手术时间、术中出血及椎间隙高度无统计学差异(P>0.05)。两组内固定物均牢靠无松动,A组有1例出现轻微排斥反应。[结论]与聚醚醚酮(PEEK)颈椎融合器相比,同种异体骨垫不但有着优越的生物弹性模量,可靠的稳定性及不可比拟的天然骨诱导性,还可以个性化匹配符合椎间隙的生理形状,在临床应用中取得了满意的融合效果,值得临床广泛推广应用。     

Estimation of renal function in diabetic nephropathy. Comparison of five methods

Plasma as well as renal clearance of 51Cr-EDTA, serum creatinine, plasma beta-2-microglobulin and endogenous creatinine clearance were compared and evaluated in patients with diabetic nephropathy and in control patients with renal disease of other origin. The difference between the plasma clearance and the renal clearance of 51Cr-EDTA, that is the extrarenal clearance, was found to be higher in diabetics than in control patients (7.0 vs. 3.5 ml/min; p less than 0.001). The serum creatinine correlated well with the glomerular filtration rate (GFR), but in the individual case the GFR was not at all predictable from serum creatinine. The plasma beta-2-microglobulin did not correlate better than serum creatinine to 51Cr-EDTA clearance, and did not permit an earlier diagnosis of renal insufficiency. Endogenous creatinine clearance overestimated GFR by 0-180%. Due to residual urine, the coefficient of variation was higher in diabetic patients than in controls, but the effect of this imperfection was reduced by using multiple collection periods. In conclusion, the renal clearance of 51Cr-EDTA was found to be preferable to the other methods.