Experimental Study on Allogenic Decalcified Bone Matrix as Carrier for Bone Tissue Engineering

The biocompatibility and osteogenic activity of allogenic decalcified bone matrix (DBM) used as a carrier for bone tissue engineering were studied. Following the method described by Urist, allogenic DBM was made. In vitro, DBM and bone marrow stromal cell (BMSC) from rabbits were co-cultured for 3-7 days and subjected to HE staining, and a series of histomorphological observations were performed under phase-contrast microscopy and scanning electron microscopy (SEM). In vivo the mixture of DBM/BMSC co-cultured for 3 days was planted into one side of muscules sacrospinalis of rabbits, and the DBM without BMSC was planted into other side as control. Specimens were collected at postoperative week 1, 2 and 4, and subjected to HE staining, and observed under SEM. The results showed during culture in vitro, the BMSCs adherent to the wall of DBM grew, proliferated and had secretive activity. The in vivo experiment revealed that BMSCs and undifferentiated mesenchymal cells in the perivascular region invaded gradually and proliferated together in DBM/BMSC group, and colony-forming units of chondrocytes were found. Osteoblasts, trabecular bone and medullary cavity appeared. The inflammatory reaction around muscles almost disappeared at the second weeks. In pure DBM group, the similar changes appeared from the surface of the DBM to center, and the volume of total regenerate bones was less than the DBM/BMSC group at the same time. The results indicated that the mixture of DBM and BMSC had good biocompatibility and ectopic induced osteogenic activity.

Use of demineralized bone matrix in spinal fusion

Spinal fusion remains the gold-standard treatment for several pathological spine conditions. Although, autologous Iliac Crest Bone Grafting is considered the goldstandard graft choice to promote spinal fusion; however, it is associated with significant donor site morbidity and a limited graft quantity. Therefore, several bone graft alternatives have been developed, to augment arthrodesis. The purpose of this review is to present the results of clinical studies concerning the use of demineralized bone matrix(DBM), alone or as a composite graft, in the spinal fusion. A critical review of the English-language literature was conducted on Pubmed, using key word "demineralized bone matrix", "DBM", "spinal fusion", and "scoliosis". Results had been restricted to clinical studies. The majority of clinical trials demonstrate satisfactory fusion rates when DBM is employed as a graft extender or a graft enhancer.Limited number of prospective randomized controlled trials(4 studies), have been performed comparing DBM to autologous iliac crest bone graft in spine fusion. The majority of the clinical trials demonstrate comparable efficacy of DBM when it used as a graft extender in combination with autograft, but there is no clinical evidence to support its use as a standalone graft material. Additionally, high level of evidence studies are required, in order to optimize and clarify the indications of its use and the appropriate patient population that will benefit from DBM in spine arthrodesis.

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 andbone 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.

Induction of Bone Matrix Protein Expression by Native Bone Matrix Proteins in C2C12 Culture

Objective To study the expression of bone matrix protein （BMP） induced by bovine bone morphogenetic proteins （BMPs） in vitro. Methods Type 1 collagen, osteopontin （OPN）, osteonectin （ON）, osteocalcin （OC）, and bone sialoprotein （BSP） were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28. Results The signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the lkaline phosphatase （ALP） activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblsts in vivo and in differentiating osteoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity. Conclusion Native bovine BMP induces conversion of myoblasts into osteoblasts, produces type I collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C 12 in vitro.

The Clinical Application of Human Bone Matrix Gelatin

This paper reports the results of 24 cases of bone defect resulting from bone tumor or tumor condition excision, and of posterior spinal fusion, treated by human bone matrix gelatin. The success rate of bone defect repair and spinal fusion is 91. 67 %. The results suggest that human bone matrix gelatin has. excellent osteoinductive effect and is ideal substitute for bone autografts.

Capacity of bone induction of compound material of decalcified bone matrix combined with rhBMP-2 and impregnated with bone cement

AIM:To analyze bone inductive capacity of the compound material of decalcified bone matrix combined with rhBMP 2 and impregnated with bone cement.METHODS:To assess the experimental study, histological and Masson’s methods were used.RESULTS:The effects of compound material on the induction of bone formation were investigated in NIH mouse models.It was observed that in the with rhBMP 2 group, mesenchymal cells gathered in the implanted material at the 7th day postoperation,chondrogenesis were found at 14 to 21 days after implantation,new bone formation were observed at about 21 to 28 days after surgery and the DBM particles were absorbed by the new generated tissues gradually.CONCLUSIONS:The compound material of DBM combined with rhBMP 2 and impregnated with bone cement could induce the proliferation and migration of mesenchymal tissues that could be differentiated into cartilage and formed new bone finally.The new bone could absorb DBM particles gradually.The compound material had fair capacity of bone induction.

Constructive and biomechanical properties of material of decalcified bone matrix impregnated with bone cement

AIM:To analyze constructive and biomechanical properties of different quality ratio material impregnated decalcified bone matrix (DBM) with bone cement (BC).M ETHODS:The DBM particles and the materials impregnated 0 mg/g, 300 mg/g and 400 mg/g mass ratio DBM particles with BC were made according to the methods of Uris t et al.The compound material constructions were observed by scanning electron m icroscope and the biomechanical properties were measured by Instron mechanics te sting-machine.RESULTS:The DBM particles with irregular gaps existing within int erspace were connected with BC by the multipoint mode in the compound material. The ultimate compressive strength were 0 mg/g DBM in (59.3±2.2) MPa, 300 mg/g i n (27.1±1.8) MPa, 400 mg/g in (19.3±1.6) MPa.The ultimate bending strength wer e 0 mg/g in (54.3±3.7) MPa, 300 mg/g in (18.5±1.1) MPa, 400 mg/g in (13.3±1.4 ) MPa.CONCLUSION:The materials of DBM impregnated with BC had perfect plastic pr operty with much more irregular gaps existing within interspace.The materials co uld provide abundant biomechanical support.

Expression of Matrix Metalloproteinase-9 mRNA in Osteoporotic Bone Tissues

Matrix metalloproteinases (MMPs), a sort of irnportant enzymes involved in extracellular matrix metabolism, play critical r0Ies in the process of tissues remodeling, wound healing and metastasis of tumors. Dot blot and in situ hybridization were used in this study to detect the expression and localization of MMP- 9, an important proteolytic enzyme implicated in bone resorption, in bone tissues. The results showed that the level of MMP-9 mRNA expression in osteoporotic bone tissues was significantly higher than that in normal control group and the cell types that expressed MMP-9 mRNA incIuded mono- and multi-nuclear osteoclasts and some lining cells on the surface of bone matrix. It was suggested that MMP-9 play a key role in the development of bone loss in osteoporosis.

Soft tissue swelling incidence using demineralized bone matrix in the outpatient setting

AIM To assess use of demineralized bone matrix(DBM) use in anterior cervical discectomy and fusion(ACDF) in outpatient setting.METHODS One hundred and forty-five patients with prospectively collected data undergoing single and two level ACDF with DBM packed within and anterior to polyetheretherketone(PEEK) cages. Two groups created, Group 1(75) outpatients and control Group 2(70) hospital patients. Prevertebral soft tissue swelling(PVSTS) was measured anterior to C2 and C6 on plain lateral cervical radiographs preoperatively and one week postoperatively and fusion assessed at two years. RESULTS There was no intergroup significance between preoperative and postoperative visual analogue scales(VAS)and neck disability index(NDI) scores between Group 1 and 2. Mean preoperative PVSTS in Group 1 was 4.7 ± 0.2 mm at C2 level and 11.1 ± 0.5 at C6 level compared to Group 2 mean PVSTS of 4.5 ± 0.5 mm and 12.8 ± 0.5, P = 0.172 and 0.127 respectively. There was no radiographic or clinical evidence of adverse reaction noted. In Group 1 mean postoperative PVSTS was 5.5 ± 0.4 mm at C2 and 14.9 ± 0.6 mm at C6 compared Group 2 mean PVSTS was 4.9 ± 0.3 mm at C2 and 14.8 ± 0.5 mm at C6, P = 0.212 and 0.946 respectively. No significant increase in prevertebral soft tissue space at C2 and C6 level demonstrated.CONCLUSION ACDF with adjunct DBM packed PEEK cages showed a statistical significant intragroup improvement in VAS neck pain scores and NDI scores(P = 0.001). There were no reported serious patient complications; post-operative radiographs demonstrated no significant difference in prevertebral space. We conclude that ACDF with DBMpacked PEEK cages can be safely done in an ASC with satisfactory outcomes.

A Comparable Study of Combinational Regenerative Therapies Comprising Enamel Matrix Derivative plus Deproteinized Bovine Bone Mineral with or without Collagen Membrane in Periodontitis Patients with Intrabony Defects

Aim: The aim of the present study was to examine the effectiveness of collagen membrane (CM) in regenerative therapy with deproteinized bovine bone mineral (DBBM) and enamel matrix derivative (EMD) for periodontal intrabony defects. Methods: Eighteen periodontal intrabony defects of nine chronic periodontitis patients were evaluated. Two defects per patient with probing pocket depth (PPD) ≥ 6 mm were assigned to two different types of treatments: EMD + DBBM + CM or EMD + DBBM. Clinical parameters including Gingival Index (GI), PPD, clinical attachment level (CAL), gingival recession (GR), bleeding on probing (BOP), tooth mobility (MOB), and the filled bone volume/rate (FBV/FBR), which was measured by cone beam computed tomography, were compared at baseline and 12 months post-treatment. Differences between groups were determined by the chisquare test, McNemar’s test, and Wilcoxon signed-rank test. Results: Clinically, PPD, CAL, and FBR significantly improved in both groups (p Conclusion: Periodontal regenerative therapies comprising EMD and DBBM with and without CM resulted in positive clinical outcomes. The use of CM may result in better outcomes in MOB decrease;however, long-term prognosis must be further studied.

TISSUE-ENGINEERED GRAFT CONSTRUCTED BY SELF-DERIVED BONE MARROW MONONUCLEAR CELLS AND HETEROGENEOUS ACELLULARIZED TISSUE MATRIX

Objective To create a method for constructing a tissue-engineered graft with self-derived bone marrow cells and heterogeneous acellular matrix.Methods The mononuclear cells were isolated from bone marrows drawn from piglets and cultured in different mediums including either vascular endothelial growth factor(VEGF)or platelet derived growth factor BB(PDGF-BB)to observe their expansion and differentiation.The aortas harvested from canines were processed by a multi-step decellularizing technique to erase.The bone marrow mononuclear cells cultured in the mediums without any growth factors were seeded to the acellular matrix.The cells-seeded grafts were incubated in vitro for 6 d and then implanted to the cells-donated piglets to substitute parts of their native pulmonary arteries.Results After 4 d culturing,the cells incubated in the medium including VEGF showed morphological feature of endothelial cells(ECs)and were positive to ECs-specific monoclonal antibodies of CD31,FLK-1,VE-Cadherin and vWF.The cells incubated in the medium including PDGF-BB showed morphological feature of smooth muscle cells(SMCs)and were positive to SMCs-specific monoclonal antibodies of α-SMA and Calponin.One hundred days after implantation of seeded grafts,the inner surfaces of explants were smooth without thrombosis,calcification and aneurysm.Under the microscopy,plenty of growing cells could be seen and elastic and collagen fibers were abundant.Conclusion Mesenchymal stem cells might exist in mononuclear cells isolated from bone marrow.They would differentiate into endothelial cells or smooth muscle cells in proper in vitro or in vivo environments.The bone marrow mononuclear cells might be a choice of seeding cells in constructing tissue-engineered graft.

Nanofibrous Scaffold Containing Osteoblast-Derived Extracellular Matrix for the Proliferation of Bone Marrow Mesenchymal Stem Cells

Extracellular matrix( ECM) plays a prominent role in establishing and maintaining an appropriate microenvironment for tissue regeneration. The aims of this study were to construct a tissue engineered scaffold by reconstituting osteoblast cell-derived ECM( O-ECM) on the electrospun nanofibrous scaffold,and further to evaluate its subsequent application for promoting the proliferation of bone marrow mesenchymal stem cells( BMSCs). To engineer a biomimetic scaffold, calvarial osteoblasts and electrospun poly-llactic acid( PLLA) nanofibers were prepared and subjected to decellularize for O-ECM deposition. To evaluate and characterize the O-ECM/PLLA scaffold, the morphology was examined and several specific mark proteins of osteoblasts matrix were evaluated.Furthermore,the cell counting kit-8( CCK-8) assay was used to detect the proliferation of the BMSCs cultivated on the O-ECM/PLLA scaffold. The results indicated O-ECM/PLLA scaffold was loaded with Collagen I, Fibronectin, and Laminin, as the composition of the marrow ECM. After decellularization,O-ECM deposition was observed in O-ECM/PLLA scaffold. Moreover,the O-ECM/PLLA scaffold could significantly enhance the proliferation of BMSCs,suggesting better cytocompatibility compared to the other groups tested. Taken together,a biomimetic scaffold based on the joint use of O-ECM and PLLA biomaterials,which represents a promising approach to bone tissue engineering, facilitates the expansion of BMSCs in vitro.

保留反应性软组织联合脱矿牙本质基质应用于拔牙位点保存

背景:反应性软组织内的干细胞间充质具有促进组织再生的潜力,脱矿牙本质基质具有良好的生物相容性,可以作为位点保存术的支架材料,促进骨细胞的附着、增殖和分化。目的:影像学分析拔牙保留反应性软组织1个月后、行脱矿牙本质基质位点保存术后6个月牙槽骨高度和宽度的变化。方法:纳入38例患者共62个拔牙位点,拔除患牙保留反应性软组织1个月后应用脱矿牙本质基质行位点保存术,术前、术后即刻、术后6个月拍摄锥形束CT,分别测量近中骨高度、中央骨高度、远中骨高度、颊侧骨高度、舌侧骨高度、牙槽嵴宽度,根据拔牙后剩余牙槽窝骨壁数量分为一壁骨缺损、二壁骨缺损、三壁骨缺损、四壁骨缺损。比较位点保存术前、术后即刻、术后6个月的骨量变化。结果与结论:①在骨愈合期间无位点发生创口感染;②与位点保存术前比较,术后即刻骨高度、骨宽度明显增加(P<0.05);术后6个月骨高度增加(P<0.05),骨宽度无明显变化(P>0.05);与术后即刻比较,术后6个月骨宽度吸收了(1.253±2.896)mm(P<0.05),骨高度无明显变化(P>0.05);③位点保存术后即刻、术后6个月4种骨缺损类型的骨高度明显增加(P<0.05),骨宽度无显著变化(P>0.05);术后6个月较术前一壁骨缺损近中骨量增长最少(P<0.05),二壁骨缺损近中骨量增长最多(P<0.05);④结果表明:脱矿牙本质基质应用于位点保存术可有效预防和减缓拔牙术后的牙槽骨吸收,能够在一定程度上重建已发生吸收的牙槽骨轮廓;拔牙术后保留反应性组织应用于脱矿牙本质基质位点保存术中,可实现创口封闭,临床疗效好;脱矿牙本质基质应用于一壁、二壁、三壁、四壁牙槽窝位点保存效果相似,但总体而言,脱矿牙本质基质应用于骨壁完整的拔牙窝时位点保存效果更为优良。

羊膜细胞外基质与膀胱细胞外基质材料修复大鼠子宫内膜损伤

背景:大量研究已证实,羊膜细胞外基质与膀胱细胞外基质材料均可作为干细胞载体用于子宫内膜损伤的治疗,但是有关两种材料的比较研究相对少见。目的:对比羊膜细胞外基质与膀胱细胞外基质材料作为干细胞载体治疗子宫内膜损伤的差异。方法:采用全骨髓贴壁法分离纯化SD大鼠骨髓间充质干细胞。分别制备SD大鼠羊膜细胞外基质与膀胱细胞外基质材料,然后将骨髓间充质干细胞分别接种于两种材料表面,检测细胞增殖与黏附情况。将40只SD大鼠随机分为4组(n=10),除假手术组外,子宫内膜损伤组、羊膜细胞外基质组、膀胱细胞外基质组均通过机械干预的方式建立子宫内膜损伤模型,分别将羊膜细胞外基质/骨髓间充质干细胞复合物、膀胱基质细胞外基质/骨髓间充质干细胞复合物移植至羊膜细胞外基质组、膀胱细胞外基质组大鼠损伤内膜部位,移植后14,28 d取材检测,苏木精-伊红染色观察大鼠子宫内膜组织形态,酶联免疫吸附法分析子宫内膜组织中碱性成纤维细胞生长因子、胰岛素样生长因子1与血管内皮生长因子水平,免疫组化染色分析子宫内膜组织中波形蛋白与CD34表达。结果与结论:(1)两种细胞外基质材料均有利骨髓间充质干细胞的增殖,相较于膀胱细胞外基质材料,羊膜细胞外基质材料可促进骨髓间充质干细胞的黏附;(2)相较于假手术组,子宫内膜损伤组碱性成纤维细胞生长因子、胰岛素样生长因子1与血管内皮生长因子水平降低(P <0.01),子宫内膜组织形态发育不良,内膜厚度与腺体数量减少,波形蛋白与CD34阳性表达减少(P <0.01);相较于子宫内膜损伤组,羊膜细胞外基质组、膀胱细胞外基质组碱性成纤维细胞生长因子、胰岛素样生长因子1与血管内皮生长因子水平均升高(P <0.05或P <0.01),子宫内膜组织形态明显改善,内膜厚度与腺体数量增加,波形蛋白与CD34阳性表达增加(P <0.05或P <0.01),并且羊膜细胞外基质组的改善作用优于膀胱细胞外基质组(P <0.05);(3)结果表明,相较于膀胱细胞外基质材料,羊膜细胞外基质材料作为骨髓间充质干细胞的载体可进一步促进损伤子宫内膜的修复。

Dynamic culture improves MSC adhesion on freeze-dried bone as a scaffold for bone engineering

AIM: To investigate the interaction between mesenchymal stem cells (MSCs) and bone grafts using two different cultivation methods: static and dynamic. METHODS: MSCs were isolated from rat bone marrow. MSC culture was analyzed according to the morphology, cell differentiation potential, and surface molecular markers. Before cell culture, freeze-dried bone (FDB) was maintained in culture for 3 d in order to verify culture medium pH. MSCs were co-cultured with FDB using two different cultivation methods: static co-culture (two-dimensional) and dynamic co-culture (three-dimensional). After 24 h of cultivation by dynamic or static methods, histological analysis of Cell adhesion on FDB was performed. Cell viability was assessed by the Trypan Blue exclusion method on days 0, 3 and 6 after dynamic or static culture. Adherent cells were detached from FDB surface, stained with Trypan Blue, and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture. Statistical analyses were performed with SPSS and a P < 0.05 was considered significant. RESULTS: The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures. Rat MSCs were positive for CD44, CD90 and CD29 and negative for CD34, CD45 and CD11bc. FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH (P > 0.05). In histological analysis, there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods (P < 0.05). The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method. On day 0, the cell viability in the dynamic system was significantly higher than in the static system (P < 0.05). There was a statistical difference in cell viability between days 0, 3 and 6 after dynamic culture (P < 0.05). In static culture, cell viability on day 6 was significantly lower than on day 3 and 0 (P < 0.05). CONCLUSION: An alternative cultivation method was developed to improve the MSCs adhesion on FDB, demonstrating that dynamic co-culture provides a superior environment over static conditions.

Reconstruction of Mandibular Defect with Porous β-TCP/BMG Artificial Bone

The purpose of this study is to explore the feasibility of repairing mandibular defect with osteoconductive porous beta-tricalcium phosphate （ β- TCP ） combined with osteoinductive bone matrix gelatin （ BMG ）, which has good physical property and biompatibility and maybe a new type of artificial bone substitute. Bilateral mandible defects sized 1.0 cm ×0. 6 cm×0. 3 cm of fifteen rabbits were implanted with β- TCP （ control group ） and β- TCPI BMG（ experimental group ） respectively. The specimens were harvested at 4^th , 8^th and 12^th weeks post-operation and examined by gross, X-ray, histomorphological and the computer analysis. The volume of newly-formed bone of the experimental side was much much than that of the control side at 4^th , 8^th and 12^th weeks. There is significant difference （p 〈 0.05） between experimental group and control group. Computer-aided X- ray analysis showed that the density of newly-formed bone, compared with the control side, were similar to the surrounding natural bone at 12^th week after operntion. Therefore, β- TCP/ BMG is a better bone substitute than TCP and is a degradable and biocompntible artificial bone substitute material and is osteoinductive and osteoconductive .

Smart scaffolds in bone tissue engineering: A systematic review of literature

AIM: To improve osteogenic differentiation and attachment of cells.METHODS: An electronic search was conducted inPub Med from January 2004 to December 2013. Studies which performed smart modifications on conventional bone scaffold materials were included. Scaffolds with controlled release or encapsulation of bioactive molecules were not included. Experiments which did not investigate response of cells toward the scaffold(cell attachment, proliferation or osteoblastic differentiation) were excluded. RESULTS: Among 1458 studies, 38 met the inclusion and exclusion criteria. The main scaffold varied extensively among the included studies. Smart modifications included addition of growth factors(group Ⅰ-11 studies), extracellular matrix-like molecules(group Ⅱ-13 studies) and nanoparticles(nano-HA)(group Ⅲ-17 studies). In all groups, surface coating was the most commonly applied approach for smart modification of scaffolds. In group I, bone morphogenetic proteins were mainly used as growth factor stabilized on polycaprolactone(PCL). In group Ⅱ, collagen 1 in combination with PCL, hydroxyapatite(HA) and tricalcium phosphate were the most frequent scaffolds used. In the third group, nano-HA with PCL and chitosan were used the most. As variable methods were used, a thorough and comprehensible compare between the results and approaches was unattainable.CONCLUSION: Regarding the variability in methodology of these in vitro studies it was demonstrated that smart modification of scaffolds can improve tissue properties.

Bovine Bone Charcoal as Support Material for Immobilization of <em>Bacillus firmus</em>Strain 37 and Production of Cyclomaltodextrin Glucanotransferase by Batch Fermentation in a Fluidized Bed

The process described in the present work uses air supplementation in a fluidized bed reactor containing Bacillus firmus strain 37 immobilized on active bovine bone charcoal, to produce by batch fermentation the enzyme CGTase (cyclomaltodextrin-glucanotransferase). Three different aeration rates were evaluated. The maximum CGTase activity was achieved after 120 hours of fermentation with aeration rate of 2 vvm and was equal to 2.48 U/mL. When 0.5 and 1 vvm were used the enzymatic activities achieved 1.1 and 0.57 U/mL, respectively. Bovine bone charcoal was characterized in terms of surface area, pore size and volume. To the best of our knowledge, the immobilization of microorganism cells in bovine bone charcoal for CGTase production has not been reported in the literature. Our results showed that fluidized bed reactor allows retaining high concentration of biomass, improving biomass-substrate contact and operation at low residence times, which resulted in improved enzyme production. Therefore, the process as proposed has great potential for industrial development.