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.

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.

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.

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.

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.

Development of photoreactive demineralized bone matrix 3D printing colloidal inks for bone tissue engineering

Demineralized bone matrix(DBM)has been widely used clinically for dental,craniofacial and skeletal bone repair,as an osteoinductive and osteoconductive material.3D printing(3DP)enables the creation of bone tissue engineering scaffolds with complex geometries and porosity.Photoreactive methacryloylated gelatin nanoparticles(GNP-MAs)3DP inks have been developed,which display gel-like behavior for high print fidelity and are capable of post-printing photocrosslinking for control of scaffold swelling and degradation.Here,novel DBM nanoparticles(DBM-NPs,∼400 nm)were fabricated and characterized prior to incorporation in 3DP inks.The objectives of this study were to determine how these DBM-NPs would influence the printability of composite colloidal 3DP inks,assess the impact of ultraviolet(UV)crosslinking on 3DP scaffold swelling and degradation and evaluate the osteogenic potential of DBM-NP-containing composite colloidal scaffolds.The addition of methacryloylated DBM-NPs(DBM-NP-MAs)to composite colloidal inks(100:0,95:5 and 75:25 GNP-MA:DBM-NP-MA)did not significantly impact the rheological properties associated with printability,such as viscosity and shear recovery or photocrosslinking.UV crosslinking with a UV dosage of 3 J/cm2 directly impacted the rate of 3DP scaffold swelling for all GNP-MA:DBM-NP-MA ratios with an∼40%greater increase in scaffold area and pore area in uncrosslinked versus photocrosslinked scaffolds over 21 days in phosphate-buffered saline(PBS).Likewise,degradation(hydrolytic and enzymatic)over 21 days for all DBM-NP-MA content groups was significantly decreased,∼45%less in PBS and collagenase-containing PBS,in UV-crosslinked versus uncrosslinked groups.The incorporation of DBM-NP-MAs into scaffolds decreased mass loss compared to GNP-MA-only scaffolds during collagenase degradation.An in vitro osteogenic study with bone marrow-derived mesenchymal stem cells demonstrated osteoconductive properties of 3DP scaffolds for the DBM-NP-MA contents examined.The creation of photoreactive DBM-NP-MAs and their application in 3DP provide a platform for the development of ECM-derived colloidal materials and tailored control of biochemical cue presentation with broad tissue engineering applications.

Nanostructure and mechanical properties of the osteocyte lacunar-canalicular network-associated bone matrix revealed by quantitative nanomechanical mapping

Osteocytes are the main bone cells embedded in the bone matrix where they form a large surface-area network called the lacunar-canalicular network （LCN）, interconnecting their resident spaces with the lacunae by the canaliculi. Increasing evidence points toward osteocytes playing a pivotal role in maintaining bone quality. On the one hand, osteocytes transmit mechanical strain and microenvironmental signals through the LCN to regulate the activity of osteoblasts and osteoclasts; on the other hand, osteocytes are suggested to be able to remodel the LCN-associated bone matrix. However, due to the challenges involved in the assessment and characterization of the LCN-associated bone matrix, little is known about its structure and the corresponding mechanical properties. In this work, we used quantitative nanomechanical mapping, backscattered electron imaging, and nanoindentation to characterize the LCN-associated bone matrix. The results show that the techniques can be used to probe the LCN-associated bone matrix. Nanoindentation and quantitative mechanical mapping reveal spatially inhomogeneous mechanical properties of the bone matrix associated with the osteocyte lacunae and canaliculi. The obtained nano-topography and corresponding nano-mechanical maps reveal altered mechanical properties in the immediate vicinity of the osteocyte lacunae and canaliculi, which cannot be explained solely by the topographic change.

Large-sized bone defect repair by combining a decalcified bone matrix framework and bone regeneration units based on photo-crosslinkable osteogenic microgels

Physiological repair of large-sized bone defects is great challenging in clinic due to a lack of ideal grafts suitable for bone regeneration.Decalcified bone matrix(DBM)is considered as an ideal bone regeneration scaffold,but low cell seeding efficiency and a poor osteoinductive microenvironment greatly restrict its application in large-sized bone regeneration.To address these problems,we proposed a novel strategy of bone regeneration units(BRUs)based on microgels produced by photo-crosslinkable and microfluidic techniques,containing both the osteogenic ingredient DBM and vascular endothelial growth factor(VEGF)for accurate biomimic of an osteoinductive microenvironment.The physicochemical properties of microgels could be precisely controlled and the microgels effectively promoted adhesion,proliferation,and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in vitro.BRUs were successfully constructed by seeding BMSCs onto microgels,which achieved reliable bone regeneration in vivo.Finally,by integrating the advantages of BRUs in bone regeneration and the advantages of DBM scaffolds in 3D morphology and mechanical strength,a BRU-loaded DBM framework successfully regenerated bone tissue with the desired 3D morphology and effectively repaired a large-sized bone defect of rabbit tibia.The current study developed an ideal bone biomimetic microcarrier and provided a novel strategy for bone regeneration and large-sized bone defect repair.

Effect of cefazolin loaded bone matrix gelatin on repairing large segmental bone defects and preventing infection

Objective: To explore the possibility of repairi ng long segmental bone defects and preventing infection with cefazolin loaded bo ne matrix gelatin (C-BMG).Methods: C-BMG was made from putting cefazolin into BMG by vac uum absorption and lyophilization techniques. The sustaining period of effective drug concentration in vitro and in vivo was detected. The time of inhibiting ba cteria,and the drug concentration in local tissues (bone and muscle) and plasma after implantation of C-BMG were examined by high performance liquid chromatog raphy. Results: The effective inhibition time to staphylococcus aureus of C-BMG was 22 days in vitro; while 14 days in vivo. The cefazolin concentrat ion in local tissues was higher in early stage,and later it kept a stable and l ow drug release. C-BMG showed an excellent ability to repair segmental long bon e defects.Conclusions: C-BMG can gradually release cefazolin with effect ive drug concentration and has excellent ability to repair segmental bone defect s. It can be used to repair segmental long bone defects and prevent infection af ter operation.

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×3 mm×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 ofhBMSCs was evaluated on days 1, 3, 5, 7 and 14. The concentration of alkaline phosphatase （ALP）, osteocalcin （OC）, and Ca^2＋ 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）, Ca^2＋ （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.

An overview on bone protein extract as the new generation of demineralized bone matrix

Bone protein extract is regarded as the new generation of demineralized bone matrix. The aim of this paper is to describe and characterize the properties of demineralized bone matrix and its new generation product in addition to its application in animal and human studies. Bone protein extract has features of osteoconductivity, osteoinductivity and osteogenicity, which originate from its unique and precise processing. It has exhibited powerful bone formation capacity both in animal experiments and in clinical trials by providing an optimal microenvironment for osteogenesis. Furthermore, not only does it have excellent bio- compatibility, it also has good compatibility with other implant materials, helping it bridge the host and implanted materials. Bone protein extract could be a promising alternative for demineralized bone matrix as a bone graft substitute.

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.

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.