Repair of large segmental bone defects using bone marrow stromal cells with demineralized bone matrix

Objective: The aim of the present study was to evaluate the effect of tissue‐engineered constructs on repair of large segmental bone defects in goats. Methods: Allogenic demineralized bone matrix (aDBM) was seeded with autologous marrow stromal cells (aMSC) for seven days to construct DBM–MSC grafts prior to implantation. 24 goats were randomly divided into three groups (eight in each). In each group, 3 cm diaphyseal femoral defects were created unilaterally, and subsequently filled with the DBM‐MSC grafts, DBM alone and an untreated control, respectively. Radiological analysis and biomechanical evaluation were performed at 12 and 24 weeks after operation. Results: Obvious increases in radiological scoring and biomechanical strength were found in the DBM‐MSC group when compared to the DBM group. X‐ray examination showed excellent bone healing in the DBM‐MSC group, whereas only partial bone repair was seen in the DBM group, and no healing in untreated controls. Histologically, a tendency to bone regeneration and remodeling was far more obvious for the DBM‐MSC group than the DBM only and untreated controls. Conclusion: Our results strongly suggest that transplantation of bone MSC within a DBM could have advantages for the bone repair of large segmental defects.     

The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats.

Subcutaneous implants of a recombinant human form of the bone-inducing protein rhBMP-2 (recombinant human bone morphogenetic protein-2) in rats have resulted in the local induction of endochondral bone formation. To test the osteoinductive activity of rhBMP-2 in an osseous location, we created five-millimeter segmental defects in the femora of forty-five adult male Sprague-Dawley rats. Two doses of lyophilized rhBMP-2 (1.4 or 11.0 micrograms) were implanted in each defect, together with guanidine-hydrochloride extracted demineralized rat-bone matrix as a carrier, and the results were compared with those in rats that had implantation of guanidine-hydrochloride extracted demineralized rat-bone matrix only. The formation and healing of bone were determined by radiographic, histological, and mechanical analysis. Both doses of rhBMP-2 induced formation of endochondral bone in the osseous defects in a dose-related manner. Implantation of 11.0 micrograms of rhBMP-2 yielded significant (p less than 0.05) bone formation, resulting in radiographic, histological, and mechanical evidence of union. Despite new-bone formation in the defects that had received 1.4 micrograms of rhBMP-2, no instances of union were observed.     

Regeneration of calvarial defects by a composite of bioerodible polyorthoester and demineralized bone in rats.

A study was performed to evaluate regeneration of defects in rat calvaria either unfilled or filled with a bioerodible polyorthoester only, demineralized bone only, or a composite of both. At 4 weeks, histological and radiographic studies showed that defects filled with a composite of bioerodible polyorthoester and demineralized bone or demineralized bone alone were bridged by bone. Unfilled defects or defects filled with polyorthoester only did not heal. The polyorthoester caused slight inflammation that subsided by 3 weeks, and only traces of the filler could be detected at 4 weeks. The polyorthoester provided local hemostasis when used either alone or in composites with demineralized bone. The composite implant was moldable, easily contoured, and technically easier to use than demineralized bone alone.     

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.     

Accelerated repair of cortical bone defects using a synthetic extracellular matrix to deliver human demineralized bone matrix.

Injectable hydrogel and porous sponge formulations of Carbylan-GSX, a crosslinked synthetic extracellular matrix (ECM), were used to deliver human demineralized bone matrix (DBM) in a rat femoral defect model. A cortical, full-thickness 5-mm defect was created in two femurs of each rat. Six rats were assigned to each of five experimental groups (thus, 12 defects per group). The defects were either untreated or filled with Carbylan-GSX hydrogel or sponges with or without 20% (w/v) DBM. Radiographs were obtained on day 1 and at weeks 2, 4, 6, and 8 postsurgery of each femur. Animals were sacrificed at week 8 postsurgery and each femur was fixed, embedded, sectioned, and processed for Masson's Trichrome staining. The bone defects were measured from radiographs and the fraction of bone healing was calculated. The average fractions of bone healing for each group were statistically different among all groups, and all treatment groups were significantly better than the control group. The Carbylan-GSX sponge with DBM was superior to the sponge without DBM and to the hydrogel with DBM. Histology showed that defects treated with the Carbylan-GSX sponge plus DBM were completely filled with newly generated bone tissue with a thickness comparable to native bone. Carbylan-GSX sponge was an optimal delivery vehicle for human DBM to accelerate bone healing.     

Repair of long bone defects with demineralized bone matrix and autogenous bone composite

Background:

Repair of diaphyseal bone defects is a challenging problem for orthopedic surgeons. In large bone defects the quantity of harvested autogenous bone may not be sufficient to fill the gap and then the use of synthetic or allogenic grafts along with autogenous bone becomes mandatory to achieve compact filling. Finding the optimal graft mixture for treatment of large diaphyseal defects is an important goal in contemporary orthopedics and this was the main focus of this study. The aim of this study is to investigate the efficacy of demineralized bone matrix (DBM) and autogenous cancellous bone (ACB) graft composite in a rabbit bilateral ulna segmental defect model.

Materials and Methods:

Twenty-seven adult female rabbits were divided into five groups. A two-centimeter piece of long bone on the midshaft of the ulna was osteotomized and removed from the rabbits’ forearms. In group 1 (n=7) the defects were treated with ACB, in group 2 (n=7) with DBM, and in group 3 (n=7) with ACB and DBM in the ratio of 1:1. Groups 4 and 5, with three rabbits in each group, were the negative and positive controls, respectively. Twelve weeks after implantation the rabbits were sacrificed and union was evaluated with radiograph (Faxitron), dual-energy x-ray absorptiometry (DEXA), and histological methods (decalcified sectioning).

Results:

Union rates and the volume of new bone in the different groups were as follows: group 1 - 92.8% union and 78.6% new bone; group 2 - 72.2% union and 63.6% new bone; and group 3 - 100% union and 100% new bone. DEXA results (bone mineral density [BMD]) were as follows: group 1 - 0.164 g/cm², group 2 - 0.138 g/cm², and group 3 - 0.194 g/cm².

Conclusions:

DBM serves as a graft extender or enhancer for autogenous graft and decreases the need of autogenous bone graft in the treatment of bone defects. In this study, the DBM and ACB composite facilitated the healing process. The union rate was better with the combination than with the use of any one of these grafts alone.     

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     

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.     

Mechanisms of action of demineralized bone matrix in the repair of cortical bone defects

Demineralized bone matrix commonly is used to enhance and to facilitate bone grafting after skeletal injury or disease; however, the biologic bases for its bone-inducing abilities remain obscure. We have taken advantage of a mouse model of cortical bone defect healing to elucidate its mechanisms of action in vivo. Demineralized bone matrix combined with hyaluronan improved skeletal healing by inducing early deposition of an osteoid matrix. Demineralized bone matrix combined with hyaluronan might accelerate bone formation because it serves as a scaffold on which osteoprogenitor cells attach. We tested this possibility by comparing demineralized bone matrix combined with hyaluronan with heat-inactivated demineralized bone matrix combined with hyaluronan and found that the intact material was superior in terms of its ability to stimulate new bone formation. We also compared the bone inducing capacity of demineralized bone matrix combined with hyaluronan with a synthetic collagen sponge and found that not only the synthetic collagen scaffold delayed bone healing but also impaired bony bridging at later stages of repair. Another important property of demineralized bone matrix combined with hyaluronan was its ability to become actively degraded by osteoclasts during healing. Therefore, demineralized bone matrix combined with hyaluronan may not only attract osteoblasts and stimulate their differentiation, but also induce bone matrix resorption, which is a critically important regulator of bone formation and mineralization.     

Induction of bone by a demineralized bone matrix gel: A study in a rat femoral defect model

Demineralized bone matrix contains osteoinductive factors and stimulates filling of gaps and defects with bone; however, it is difficult to handle by itself and various preparations have been tested. Demineralized bone matrix with a gel consistency now is available for clinical use. We studied, in a femoral segment defect in the rat, the effects of rat demineralized bone matrix gel with and without a ceramic substratum. This preparation is analogous to the human demineralized bone matrix in the same carrier, used clinically for humans. One hundred adult male Fischer rats were divided into 10 experimental groups. Independent variables included the presence or absence of hydroxyapatite ceramic cylinders, the presence of demineralized bone matrix in carrier or carrier alone (glycerol), and the duration of observation (1, 2 and 4 months). Defects filled with the gel alone had significantly higher radiographic scores for host-graft union at 4 months compared with ceramic with the gel, ceramic alone, or carrier alone. Demineralized bone matrix gel significantly increased the total histologic score for host-graft union, whether ceramic was present or not, and a three-way interaction occurred among ceramic, the gel, and time. Demineralized bone matrix gel was an effective inducer of bone formation in this model. An additional substratum was not required; in fact, significantly more bone was formed in the absence of the ceramic cylinder. Neither the gel nor the ceramic were impediments to revascularization of the defect. Host-graft union was enhanced by demineralized bone matrix gel but not by the ceramic cylinder.     

The use of demineralized bone matrix for anterior cruciate ligament reconstruction: a radiographic,histologic, and immunohistochemical study in rabbits

Tendon-bone healing is crucial in success of anterior cruciate ligament (ACL) reconstruction surgery. Demineralized bone matrix (DBM) is a physiological component that has the inherent potential of bone regeneration. We hypothesized that the alternative bone substitute can affect the structural properties of tendon graft in tibial tunnel healing. Five 12-week-old New Zealand white rabbits in study group underwent unilateral ACL reconstructions plus the application of 0.5 cc DBM in the tibial tunnel. The assessment included radiological assessment and histologic and immunohistochemical analyses. Radiological examination revealed that DBM group had the least displacement of tendon in tibial tunnel (0.4 ± 0.12; P = 0.03). Histologic examination showed significantly better integration between tendon and bone in DBM group (77.62 ± 2.08; P = 0.001). On immunohistochemical analysis, the DBM group showed significantly higher expressions of bone morphogenetic protein-2 and vascular endothelial growth factor than control group (51.98 ± 3.02, 84.06 ± 1.86; P = 0.001, P < 0.001). DBM enhances the tendon-bone healing in ACL reconstruction. DBM has the potential use in ACL surgery.     

Porous ceramics as bone graft substitutes in long bone defects: A biomechanical,histological, and radiographic analysis

Three porous ceramic bone graft materials were compared with regard to their ability to heal a 2.5 cm defect created surgically in a bilateral canine radius model. The ceramic materials were analyzed at 12 and 24 weeks after surgery and included tricalcium phosphate, hydroxyapatite, and collagen hydroxyapatite, which contained a mixture of 35% tricalcium phosphate and 65% hydroxyapatite with added collagen. Each material was evaluated alone and with added bone marrow aspirate. All the implants were compared with a graft of autogenous cancellous bone in the contralateral radius. Biomechanical testing and radiographic evauation revealed that the addition of bone marrow aspirate was essential for tricalcium phosphate and hydroxyapatite to achieve results comparable with those of cancellous bone. Collagen hydroxyapatite performed well without the addition of bone marrow, although the addition of marrow did have a positive effect. Further qualitative radiographic and histological analysis demonstrated that tricalcium phosphate was the only ceramic that showed any sign of degradation at 24 weeks. This observed degradation proved to be an important factor in evaluating radiographs because the radiodensity of collagen hydroxyapatite and hydroxyapatite interfered with the determination of radiographic union. At 24 weeks, tricalcium phosphate with bone marrow was the material that performed most like cancellous bone. In this study, the biomechanical and radiographic parameters of tricalcium phosphate with bone marrow were roughly comparable with those of cancellous bone at 12 and 24 weeks. Tricalcium phosphate was the only implant that showed significant evidence of degradation at 24 weeks by both histological and radiographic evaluations, and this degradation took place only after a degree of mechanical competence necessary for weight-bearing was achieved.     

The effect of a composite of polyorthoester and demineralized bone on the healing of large segmental defects of the radius in rats.

The effect of a composite of demineralized bone mixed with polyorthoester on the healing of large segmental defects in the rat radius was studied. Sixty male Wistar rats were divided into four groups, A through D, and an osteoperiosteal diaphyseal defect of 50 per cent of the length of the bone was made in the right radius of each rat. In Group A, the defect was filled with polyorthoester and demineralized bone; in Group B, demineralized bone; and in Group C, polyorthoester. No material was implanted in the defects in the Group-D rats. The rats were killed fifty days postoperatively. The formation of bone in the defects was quantified with computer-assisted measurements of the area on radiographs. The host-tissue response was evaluated with light microscopy. Defects that had been filled with the composite of polyorthoester and demineralized bone or with demineralized bone alone showed regeneration of bone corresponding to 93.6 and 77.6 per cent of the area of the defect, respectively. Defects that had no implant or that had been filled with polyorthoester alone showed significantly less formation of bone. No inflammation was seen with light microscopy, and only traces of the polyorthoester could be detected in the defects that had been filled with the composite or with polyorthoester alone.     

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.     

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.     

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.     

Comparison of demineralized allogeneic bone matrix grafting (the Urist procedure) and the Ilizarov procedure in large diaphyseal defects in sheep

The bone inductive capability of the Urist and Ilizarov procedures was compared in the repair of large diaphyseal defects in sheep. In 30 animals, a 4 cm segmental defect was created in the middle portion of the right femur and was stabilized with an external fixator. The sheep were divided into four groups according to the type of reconstruction of the defect. In group 1, a demineralized allogeneic bone matrix (DABM) cylinder was used; in group 2, DABM chips; and in group 3, gradual transport of a piece of bone detached from the proximal femoral fragment was used to fill the defect. Group 4 served as a control (the defect was left empty). New bone formation was assessed by serial radiographs until the time of death at 2 or 4 months. Postmortem specimens were analyzed with respect to bone mineral content, uptake of isotopes (⁴⁵Ca and ³H-proline), and histology. The first signs of new bone formation were radiographically evident at 4 weeks. In the two groups in which reconstruction involved DABM (Urist procedures), new bone failed to form in eight of the 13 animals. Full bridging of the defect was observed at 8 weeks in one animal with a DABM cylinder and two with DABM chips. No decisive difference in bone yield could be demonstrated between the two Urist procedures. In the group treated with the Ilizarov procedure, new bone formation consistently occurred at a high rate; full bridging of the defect was observed in seven of the eight animals. Bone mineral scanning and histologic analysis essentially confirmed the radiographic results. Uptake of isotopes was selectively analyzed in two sheep from each experimental group in which new bone formation was exhibited in the defect; new bone formation was increased compared with that in the contralateral femur but was equal among the three experimental groups. Our study shows that gradual transport of a detached piece of autogeneic bone (Ilizarov procedure) is more effective than implantation of DABM (Urist procedure) in eliciting new bone formation in large diaphyseal defects in sheep. The variable bone induction by DABM may be explained by differences in host immune responses to the implants.     

海绵状、泥灰状脱钙骨基质修复骨缺损

目的探讨海绵状脱钙骨基质(DBM)和泥灰状DBM修复骨缺损的能力。方法将兔四肢长骨骨干脱脂、脱钙后制成长度为500～1000 μm的纤维状及直径为200～400μm的颗粒状DBM,并与2%明胶混合分别制成海绵状DBM和泥灰状DBM。在9只兔双侧桡骨中段做一长10mm的骨膜骨缺损,分别植入海绵状DBM和泥灰状DBM及空白对照,每组各6个缺损,术后观察6周,于4、6周时拍摄X线片,并对实验动物的大体标本、骨密度、生物力学、新生骨矿化率及病理组织学改变进行观察。结果术后4、6周X线片显示两实验组骨缺损均修复,骨髓腔完整;空白对照组无一例修复骨缺损。骨密度测量显示海绵状DBM组新生骨骨密度与正常桡骨间差异无显著性(P >0.05),但泥灰状DBM组的新生骨骨密度与正常桡骨间差异有显著性(P< 0.05)。术后6周生物力学测定显示海绵状DBM组新生骨极限压缩强度值与正常桡骨差异无显著性(P >0.05),泥灰状DBM组低于正常桡骨且差异有显著性(P< 0.05)。两实验组新生骨矿化率差异无显著性(P >0.05)。组织学观察显示两实验组中DBM绝大部分被吸收,形成板状骨骨小梁及完整的骨髓腔,塑形完整, 新生骨内可见骨单位及局部尚未完全骨化的新生骨。结论海绵状DBM和泥灰状DBM均具有诱导成骨活性和骨传导能力,使用方便,新生骨塑形完整,生物力学强度高,矿化     

Treatment of nonunion by percutaneous injection of bone marrow and demineralized bone matrix. An experimental study in dogs

Successful treatment of nonunited fractures remains a major clinical challenge. Because bone marrow and demineralized bone matrix (DBM) are capable of stimulating osteogenesis, experiments were designed to test the effectiveness of bone marrow or DBM or both when injected percutaneously into a canine nonunion model. Six-millimeter segmental defects were created in the midtibial diaphysis of 24 adult mongrel dogs and held distracted by external fixation. For comparative purposes, a 0.5-mm osteotomy was created in five dogs. Five weeks later, the 6-mm defects were injected with either saline, autogeneic marrow, DBM powder, a composite of bone marrow and DBM, or treated by open grafting techniques with autogenic cancellous bone. Healing of the defect was evaluated roentgenographically, biomechanically (three-point bending), histologically, and biochemically 13 weeks postsurgery. Marrow and DBM stimulated defect healing. However, the combination of bone marrow with DBM produced a synergistic response in the defect, which was greater than the sum of either marrow or DBM alone. Healing in the composite-grafted dogs was comparable to those treated by standard cancellous bone grafting. These data suggest that percutaneous injection of bone marrow and DBM may be a potential alternative that offers numerous advantages over standard open grafting techniques in the treatment of fractures with nonunited defects.