Demineralized Bone Matrix Particles Research Articles

Black Phosphorus-Based Dynamic Self-Healing Hydrogel to Integrate Demineralized Bone Matrix and Noggin-Targeting siRNA for Synergistic Osteogenesis.

Although a demineralized bone matrix (DBM) is often used as an alternative to an autologous bone graft, its clinical application is still hampered by easy dispersion of DBM particles and insufficient osteoinductivity in the defect site. Herein, we designed a self-healing hydrogel for DBM that can rapidly restore its structural integrity after damage based on amino-rich black phosphorus (BP) nanosheets and aldehyde-functionalized hyaluronic acid (AHA). Given the increased expression of bone morphogenetic protein (BMP) antagonists by DBM stimulation, the osteogenic potency of DBM in the hydrogel carrier was further enhanced by abrogating the BMP antagonism. The BP/AHA hydrogel provided dynamic polymer-nanosheet networks that combine injectability, modability, and physical stability with high DBM loading, where the BP nanosheets served as osteogenic cross-linkers to promote biomineralization and deliver siRNA to suppress undesirable expression of BMP antagonist noggin by DBM. As a result, the BP/AHA hydrogel integrated with DBM and noggin-targeting siRNA synergistically promoted osteogenic differentiation of mesenchymal stem cells by enhancing BMP/Smad signaling. This work demonstrates a promising strategy to improve the efficacy of bone regeneration using bone graft.

Self-Assembled Nanocomposite Hydrogels as Carriers for Demineralized Bone Matrix Particles and Enhanced Bone Repair.

Demineralized bone matrix (DBM) has been widely used as an allogeneic alternative to autologous bone graft for bone repair. However, more extensive use of DBM is limited due to its particulate nature after demineralization and rapid particle dispersion following irrigation, resulting in unpredictable osteoinductivity. Here, a new design of injectable hydrogel carriers for DBM that combine self-healing ability and osteogenic properties based on the self-assembly of guanidinylated hyaluronic acid and silica-rich nanoclays is reported. The nanoclays serve as reversible linkages to form a dynamic hydrogel network with the guanidine moieties on the polymer chains. Gelation kinetics and mechanical properties can be controlled by altering nanoclay content in the hydrogel. The resulting hydrogel exerts self-healing ability due to its dynamic crosslinks and well retains its overall performance with high DBM loading. The hydrogel exhibits great cytocompatibility and osteogenic effects mediated by the nanoclays. In vivo delivery of DBM using the nanocomposite hydrogel further demonstrates robust bone regeneration in a mouse calvarial defect model in comparison to DBM delivered with aqueous HA. This work suggests a promising hydrogel platform for many applications including therapeutic delivery and tissue engineering.

Enhanced osteogenicity of the demineralized bone-dermal matrix composite by the optimal partial demineralization for sustained release of bioactive molecules.

Allogenic demineralized bone matrix (DBM), processed to expose bioactive proteins imbedded by calcium salts, is widely used for bone repair and regeneration as an alternative to the autologous bone graft. However, demineralized bone matrices from tissue banks vary significantly in residual calcium content and osteogenicity for clinical bone regeneration. The present study produced DBM with various residual calcium contents by partial demineralization using ethylenediaminetetraacetic acid disodium (EDTA) and hydrochloric acid. Compositional analysis reveals that, as the percent weight loss of bone materials increases from 0% to 74.9% during demineralization, the residual calcium content of DBM decreases from 24.8% to 0.2% and collagen content increases from 29.7% to 92.6%. Calorimetrical analysis and Fourier transform infrared (FTIR) analysis demonstrated that demineralization to the residual calcium content of <4% enables the complete exposure and/or release of bone collagen fibers and other bioactive molecules. In order to evaluate the relationship between the extent of demineralization and the osteogenicity of DBM, DBM particles were fabricated with the aid of acellular dermal matrix (ADM) microfibers to form flexible foam-like DBM/ADM composites. Proteomic analysis identified various type collagens and bone formation-related bioactive molecules in both ADM and DBM. Using the rat bilateral Φ = 5 mm calvarium defect repair model, the study had shown that the DBM/ADM composite with ~20% DBM residual calcium (e.g., ~40% calcium being removed) maximized the osteogenicity for bone defect repair after 4 and 8 weeks. DBM with ~40% calcium removal had the maximal osteogenicity presumably through the sustained release of bioactive molecules during the process of bone regeneration.

Injectable demineralized bone matrix particles and their hydrogel bone grafts loaded with β-tricalcium phosphate powder and granules: A comparative study.

Demineralized bone matrix (DBM), has been used as a bone-graft material because of its osteoconductivity and osteoinductivity. However, the previous research report that supports the single use of DBM is limited by its rapid resorption caused by the lack of calcium and phosphate. β-Tricalcium phosphate (TCP) is an enriched calcium phosphate material suitable for bone healing with osteoconductive properties. In this study, we have developed injectable bone graft by the loading two kinds of TCP in DBM particles and thermo-sensitive DBM-derived hydrogel (hDBM). TCP powder (pTCP) and TCP granules (gTCP) were loaded into hDBM and DBM, respectively. The bone formation effect was investigated according to the morphological features of TCP. Residual growth factor concentrations were investigated; microstructure and morphology were characterized by SEM. In-vitro studies showed that hDBM/DBM/pTCP and hDBM/DBM/gTCP bone grafts were biocompatible and could promote osteogenesis by up-regulating the expression of Runx2 and OPN, bone-related genes. In-vivo studies using the rabbit-femur defect model revealed that the implanted hDBM/DBM/pTCP bone graft showed similar histology to that of fibrous dysplasia with the expression of CD68, whereas hDBM/DBM/gTCP showed good bone formation. Loading of gTCP in place of pTCP was noticed as an effective way to improve bone regeneration in an injectable hDBM/DBM hydrogel-based bone graft.

Evaluation of Demineralized Bone Matrix Particles Delivered by Alginate Hydrogel for a Bone Graft Substitute: An Animal Experimental Study.

BackgroundOur objective was to explore a synthetic alginate hydrogel delivery system for the delivery of demineralized bone matrix (DBM) particles for bone graft substitutes.Material/MethodsThe physiochemical properties of surface morphology, porosity measurements, in vitro degradation, equilibrium swelling, and mechanical testing of combined DBM powder and alginate in amounts of 0 mg/1 mL, 25 mg/1 mL, 50 mg/1 mL, and 100 mg/1 mL were detected. In vitro cell culture and in vivo studies using Sprague-Dawley rats were performed to evaluate the biocompatibility and osteoinductivity of DBM-alginate (ADBM) composites.ResultsDBM particles were uniformly scattered in all composites, and macro-scale pores were omnipresent. All composites showed a similar low degradation rate, with approximately 85% of weight remaining after 15 days. As the concentration of DBM particles in composites increased, degradation in collagenase and elastic modulus increased and the pore area and swelling ratio significantly decreased. No cytotoxicity of ADBM or alginate on mesenchymal stem cells (MSCs) was observed. Cell cultivation with ADBM showed greater osteogenic potential, evidenced by the upregulation of alkaline phosphatase and alizarin red staining activity and the mRNA expression level of marker genes RUNX2, OCN, OPN, and collagen I compared with the cells grown in alginate. Evaluation of ectopic bone formation revealed the osteoinductivity of the ADBM composites was significantly greater than that of DBM particles. Osteoinduction of the composites was demonstrated by a cranial defect model study.ConclusionsThe delivery of DBM particles using a synthetic alginate hydrogel carrier may be a promising approach in bone tissue engineering for bone defects.

Enhanced Osteoinductivity of Demineralized Bone Matrix with Noggin Suppression in Polymer Matrix.

Demineralized bone matrix (DBM), a potential alternative to autologous bone-graft, has been increasingly used for clinical bone repair; however, its application in larger defects isn't successful partly due to the rapid dispersion of DBM particles and relatively lower osteoinductivity. Here, a novel strategy is created to complement the osteoinductivity of DBM by incorporating DBM in biopolymer hydrogel combined with the abrogation of BMP antagonism. Combined treatment of DBM + noggin-suppression displays increased osteogenic potency of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. Injectable chitosan (MeGC)-based hydrogel with heparinization (Hep-MeGC) is further developed to localize and stabilize DBM. Noggin-suppression reveals the significant increase in osteogenesis of hBMSCs in the photopolymerizable Hep-MeGC hydrogels with the encapsulation of DBM. Moreover, the combination of DBM + noggin-suppression in the injectable Hep-MeGC hydrogel displays a robust bone healing in mouse critical-sized calvarial defects in vivo. The mechanistic analysis demonstrates that noggin-suppression increased DBM osteoinductivity by stimulating endogenous BMP/Smad signals. These results have shown promise in DBM's ability as a prominent bone grafting material while being coupled with gene editing mechanism and a localizing three-dimensional scaffold. Together, this approach poses a significant increase in the efficiency of DBM-mediated craniofacial bone repair and dental osteointegration.

Examination of the Role of Cells in Commercially Available Cellular Allografts in Spine Fusion: An in Vivo Animal Study.

Despite the extensive use of cellular bone matrices (CBMs) in spine surgery, there is little evidence to support the contribution of cells within CBMs to bone formation. The objective of this study was to determine the contribution of cells to spinal fusion by direct comparisons among viable CBMs, devitalized CBMs, and cell-free demineralized bone matrix (DBM). Three commercially available grafts were tested: a CBM containing particulate DBM (CBM-particulate), a CBM containing DBM fibers (CBM-fiber), and a cell-free product with DBM fibers only (DBM-fiber). CBMs were used in viable states (CBM-particulatev and CBM-fiberv) and devitalized (lyophilized) states (CBM-particulated and CBM-fiberd), resulting in 5 groups. Viable cell counts and bone morphogenetic protein-2 (BMP-2) content on enzyme-linked immunosorbent assay (ELISA) within each graft material were measured. A single-level posterolateral lumbar fusion was performed on 45 athymic rats with 3 lots of each product implanted into 9 animals per group. After 6 weeks, fusion was assessed using manual palpation, micro-computed tomography (μ-CT), and histological analysis. The 2 groups with viable cells were comparable with respect to cell counts, and pairwise comparisons showed no significant differences in BMP-2 content across the 5 groups. Manual palpation demonstrated fusion rates of 9 of 9 in the DBM-fiber specimens, 9 of 9 in the CBM-fiberd specimens, 8 of 9 in the CBM-fiberv specimens, and 0 of 9 in both CBM-particulate groups. The μ-CT maturity grade was significantly higher in the DBM-fiber group (2.78 ± 0.55) compared with the other groups (p < 0.0001), while none of the CBM-particulate samples demonstrated intertransverse fusion in qualitative assessments. The viable and devitalized samples in each CBM group were comparable with regard to fusion rates, bone volume fraction, μ-CT maturity grade, and histological features. The cellular component of 2 commercially available CBMs yielded no additional benefits in terms of spinal fusion. Meanwhile, the groups with a fiber-based DBM demonstrated significantly higher fusion outcomes compared with the CBM groups with particulate DBM, indicating that the DBM component is probably the key determinant of fusion. Data from the current study demonstrate that cells yielded no additional benefit in spinal fusion and emphasize the need for well-designed clinical studies on cellular graft materials.

Influence of Geometry and Architecture on the In Vivo Success of 3D-Printed Scaffolds for Spinal Fusion.

We previously developed a recombinant growth factor-free, three-dimensional (3D)-printed material comprising hydroxyapatite (HA) and demineralized bone matrix (DBM) for bone regeneration. This material has demonstrated the capacity to promote re-mineralization of the DBM particles within the scaffold struts and shows potential to promote successful spine fusion. Here, we investigate the role of geometry and architecture in osteointegration, vascularization, and facilitation of spine fusion in a preclinical model. Inks containing HA and DBM particles in a poly(lactide-co-glycolide) elastomer were 3D-printed into scaffolds with varying relative strut angles (90° vs. 45° advancing angle), macropore size (0 μm vs. 500 μm vs. 1000 μm), and strut alignment (aligned vs. offset). The following configurations were compared with scaffolds containing no macropores: 90°/500 μm/aligned, 45°/500 μm/aligned, 90°/1000 μm/aligned, 45°/1000 μm/aligned, 90°/1000 μm/offset, and 45°/1000 μm/offset. Eighty-four female Sprague-Dawley rats underwent spine fusion with bilateral placement of the various scaffold configurations (n = 12/configuration). Osteointegration and vascularization were assessed by using microComputed Tomography and histology, and spine fusion was assessed via blinded manual palpation. The 45°/1000 μm scaffolds with aligned struts achieved the highest average fusion score (1.61/2) as well as the highest osteointegration score. Both the 45°/1000 μm/aligned and 90°/1000 μm/aligned scaffolds elicited fusion rates of 100%, which was significantly greater than the 45°/500 μm/aligned iteration (p < 0.05). All porous scaffolds were fully vascularized, with blood vessels present in every macropore. Vessels were also observed extending from the native transverse process bone, through the protrusions of new bone, and into the macropores of the scaffolds. When viewed independently, scaffolds printed with relative strut angles of 45° and 90° each allowed for osteointegration sufficient to stabilize the spine at L4-L5. Within those parameters, a pore size of 500 μm or greater was generally sufficient to achieve unilateral fusion. However, our results suggest that scaffolds printed with the larger pore size and with aligned struts at an advancing angle of 45° may represent the optimal configuration to maximize osteointegration and fusion capacity. Overall, this work suggests that the HA/DBM composite scaffolds provide a conducive environment for bone regeneration as well as vascular infiltration. This technology, therefore, represents a novel, growth-factor-free biomaterial with significant potential as a bone graft substitute for use in spinal surgery. Impact statement We previously developed a recombinant growth factor-free, three-dimensional (3D)-printed composite material comprising hydroxyapatite and demineralized bone matrix for bone regeneration. Here, we identify a range of 3D geometric and architectural parameters that support the preclinical success of the scaffold, including efficient vascularization, osteointegration, and, ultimately, spinal fusion. Our results suggest that this material holds great promise as a clinically translatable biomaterial for use as a bone graft substitute in orthopedic procedures requiring bone regeneration.

The effect of gammairradiation for collagen in demineralized bone matrix particles with differences size

Objective To investigate the effects of different particle sizes on the collagen structure of demineralized bone matrix (DBM) and the effectiveness of dry ice as an irradiation protectant in the procedure of gamma irradiation. Methods DBM samples with different particle sizes (0.5-1.0 mm, 1.2-2.8 mm, 3.3-4.7 mm and 5.7-7.0 mm) were prepared, and sterilized with several doses of gamma irradiation (0 kGy, 15 kGy and 25 kGy) at room temperature. Additionally, another group of DBM samples were sterilized with 25 kGy gamma irradiation with protective agent. Changes in surface and characteristics of collagen were observed by using scanning electron microscope (SEM), Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), differential scanning calorimetry (DSC) and carbonyl content. Results The color of collagen extract indicated that oxidative damage is directly related to irradiation dose. SEM showed that the gamma irradiation caused collagen structure disorder and fiber breakage. As the irradiation doses increased, the damage area significantly increased. When the particle size increased, the damage area tended to decrease. The DSC showed that the thermal denaturation temperature of 5.7-7 mm, 3.3-4.7 mm, 1.2-2.8 mm and 0.5-1.0 mm were 142.8℃, 97.3℃,84.3℃ and 83.9℃, respectively. The differences of the thermal denaturation temperatures among the four particle sizes were statistically significant (F=0.560, P=0.650). Collagen structure was destroyed by gamma irradiation, resulting in a decrease in collagen molecular weight. While, large particle DBM had a tendency to resist radiation damage. There was a significant difference on the contents of carbonyl in collagen from same particle sizes of DBM with different irradiation dose. The carbonyl content gradually decreased with the increase of particle size, but the difference was not statistically significant (F=0.560, P=0.650). Conclusion The gamma irradiation and collagen oxidative damage have obvious dose-response relationship. With the increase of gamma irradiation dose, the degree of collagen damage increases. The sizes of DBM could affect the sensitivity of collagen to gamma irradiation. With the decrease of particle sizes, DBM particles are more susceptible to gamma irradiation damage. Additionally, dry ice, as a radiation protection agent, has a certain degree protection effect against radiation. Key words: Bone matrix; Collagen; Radiation; Gamma rays

3D-Printed Ceramic-Demineralized Bone Matrix Hyperelastic Bone Composite Scaffolds for Spinal Fusion.

Although numerous spinal biologics are commercially available, a cost-effective and safe bone graft substitute material for spine fusion has yet to be proven. In this study, "3D-Paints" containing varying volumetric ratios of hydroxyapatite (HA) and human demineralized bone matrix (DBM) in a poly(lactide-co-glycolide) elastomer were three-dimensional (3D) printed into scaffolds to promote osteointegration in rats, with an end goal of spine fusion without the need for recombinant growth factor. Spine fusion was evaluated by manual palpation, and osteointegration and de novo bone formation within scaffold struts were evaluated by laboratory and synchrotron microcomputed tomography and histology. The 3:1 HA:DBM composite achieved the highest mean fusion score and fusion rate (92%), which was significantly greater than the 3D printed DBM-only scaffold (42%). New bone was identified extending from the host transverse processes into the scaffold macropores, and osteointegration scores correlated with successful fusion. Strikingly, the combination of HA and DBM resulted in the growth of bone-like spicules within the DBM particles inside scaffold struts. These spicules were not observed in DBM-only scaffolds, suggesting that de novo spicule formation requires both HA and DBM. Collectively, our work suggests that this recombinant growth factor-free composite shows promise to overcome the limitations of currently used bone graft substitutes for spine fusion. Impact Statement Currently, there exists a no safe, yet highly effective, bone graft substitute that is well accepted for use in spine fusion procedures. With this work, we show that a three-dimensional printed scaffold containing osteoconductive hydroxyapatite and osteoinductive demineralized bone matrix that promotes new bone spicule formation, osteointegration, and successful fusion (stabilization) when implemented in a preclinical model of spine fusion. Our study suggests that this material shows promise as a recombinant growth factor-free bone graft substitute that could safely promote high rates of successful fusion and improve patient care.

Temporal and spatial immunolocalization of osteopontin in the repair of orthopaedic bone defects treated with demineralized bone matrix

Osteopontin (OPN) is the most abundant non-collagen protein in the bone matrix, where it fulfils the function of cellular adhesion and biomineralization. In the present work, the authors report the temporal and spatial localization of OPN during the repair of experimental orthopaedic bone defects treated with demineralized bone matrix (DBM) processed by the authors. 30 rabbits were used, which were given an orthopaedic bone defect of critical size in one of the radiuses, which was filled with DBM. The rabbits were euthanized in groups of 5 individuals at days 7, 15, 21, 30, 60 and 150. Histological cuts were immunomarked to establish the spatial and temporal immunomarcation of OPN. The histological cuts were observed with an optic microscope with which histological images were captured and analysed with the ImageJ software. The image analysis allowed the authors to establish the optic density (OD) and the integrated optic density (IOD). The data was analysed with the ANOVA and Fischer LSD tests. At day 7, the presence of OPN was observed only in the DBM particles, where the OD was 0.08 and the IOD was 1.64; at day 15, OPN marked different sites of collagen condensations and cells contained in the interior of the matrix. In this period the OD was 0.096 and the IOD, 9.26. At days 21 and 30, the OPN immunosignalled osteocytes, osteoblasts, osteoclasts and hypertrophic chondrocytes in the bone trabeculae adjacent to the ossification zones. At day 21 the OD was 0.17 and IOD 6.22. At day 30, the OD was 0.14 and the IOD 2.52. At days 60 and 150, OPN was evenly distributed in the new bone matrix with an OD: 0.10 and IOD: 0.48, and OD: 0.35 and IOD: 3.80, respectively. The OD and IOD showed significant differences (p<=0.05) between days 7, 15, 21 and 30; and there was no difference at days 60 and 150 (p=0.05). OPN was found in the DBM particles: it increased the optic densities at day 15 and it diminished at day 60, after which it increased the OD and IOD again until day 150. It was established that the OPN immunoexpressed during the repair process in indifferentiated cells, osteoprogenitor chondrocytes and osteoblasts. The variation of OD and IOD allowed the authors to establish that the greatest degree of immunoexpression of OPN was at day 15 after repair initiated. On the other hand, the increase registered between days 60 and 150 post treatment was due to the biomineralization of the bone matrix.

Biomimetic open porous structured core-shell microtissue with enhanced mechanical properties for bottom-up bone tissue engineering.

Background: Microtissues constructed with hydrogels promote cell expansion and specific differentiation by mimicking the microarchitecture of native tissues. However, the suboptimal mechanical property and osteogenic activity of microtissues fabricated by natural polymers need further improvement for bone reconstruction application. Core-shell designed structures are composed of an inner core part and an outer part shell, combining the characteristics of different materials, which improve the mechanical property of microtissues.Methods: A micro-stencil array chip was used to fabricate an open porous core-shell micro-scaffold consisting of gelatin as shell and demineralized bone matrix particles modified with bone morphogenetic protein-2 (BMP-2) as core. Single gelatin micro-scaffold was fabricated as a control. Rat bone marrow mesenchymal stem cells (BMSCs) were seeded on the micro-scaffolds, after which they were dynamic cultured and osteo-induced in mini-capsule bioreactors to fabricate microtissues. The physical characteristics, biocompatibility, osteo-inducing and controlled release ability of the core-shell microtissue were evaluated in vitro respectively. Then microtissues were tested in vivo via ectopic implantation and orthotopic bone implantation in rat model.Results: The Young's modulus of core-shell micro-scaffold was nearly triple that of gelatin micro-scaffold, which means the core-shell micro-scaffolds have better mechanical property. BMSCs rapidly proliferated and retained the highest viability on core-shell microtissues. The improved osteogenic potential of core-shell microtissues was evidenced by the increased calcification based on von kossa staining and osteo-relative gene expression. At 3months after transplantation, core-shell microtissue group formed the highest number of mineralized tissues in rat ectopic subcutaneous model, and displayed the largest amount of new bony tissue deposition in rat orthotopic cranial defect.Conclusion: The novel core-shell microtissue construction strategy developed may become a promising cell delivery platform for bone regeneration.

Friday, September 28, 2018 4:05 PM–5:05 PM abstracts: basic science of spinal fusion: 236. Composition of hyperelastic bone composite scaffolds affects de novo bone formation

BACKGROUND CONTEXT Bone graft extenders for spine fusion include demineralized bone matrix (DBM) and ceramic-based products, but their efficacy is insufficient to qualify them as bona fide bone graft substitutes (BGS). We previously developed a unique 3D-printable scaffold composed of synthetic hydroxyapatite particles (HA) and poly lactic-co-glycolic acid (PLGA). PURPOSE The purpose of this study is to improve the efficacy of this hyperelastic “bone” composite (HBC) by incorporating osteoinductive DBM particles. We evaluated the ability of various iterations of these HA-DBM HBC scaffolds to promote bone regeneration and spine fusion in a rat posterolateral spine fusion (PLF) model. METHODS Female Sprague Dawley rats underwent L4-L5 PLF utilizing HBC scaffolds composed of 30vol.% PLGA and a 70vol.% particles. For each group, the particle components consisted of the following vol.% ratios of HA:DBM: 1:0, 3:1, 1:1, 1:3, and 0:1. Eight weeks postoperative, spine fusion was evaluated via radiography and manual palpation. Synchrotron microCT was used to qualitatively assess bone formation within the struts of the scaffolds. Histology (Hematoxylin/Orange G/Alcian Blue and Masson's Trichrome) was used to characterize the bone growing within and around the struts and the bone-scaffold interface. RESULTS 3:1 HA:DBM HBC scaffolds had the highest fusion rate (92%), although this was only significantly higher than the 0:1 group (42% fusion; p CONCLUSIONS This study evaluated the use of 3D printed HBC scaffolds containing both HA and DBM particles as a potential BGS. Varying the proportions of HA:DBM within each scaffold resulted in only slight differences in fusion scores and rates. However, microCT showed that de novo bone spicule formation was only present within scaffold struts that contained DBM particles. We hypothesize that (1) the presence of osteoinductive DBM is key to de novo bone formation, and (2) the hydroxyapatite provides the calcium and phosphate necessary to re-mineralize the DBM particles. Future studies will aim to further improve the efficacy of this HBC as a BGS for spine fusion.

Friday, September 28, 2018 4:05 PM–5:05 PM abstracts: basic science of spinal fusion: 239. Architectural and geometric considerations in the development of a 3D-printed hyperelastic "Bone" composite scaffold as bone graft substitute for spinal arthrodesis

BACKGROUND CONTEXT Pseudarthrosis occurs in 10%-15% of patients undergoing spine fusion. We have previously developed a unique, recombinant growth factor-free bone graft substitute (BGS) consisting of poly lactic-co-glycolic acid (PLGA) and particles of hydroxyapatite (HA) and demineralized bone matrix (DBM). This 3D-printed Hyperelastic ‘Bone’ composite (HBC) promotes spinal fusion in a rat posterolateral spinal fusion (PLF) model at rates ranging from 58% to 92%. PURPOSE The purpose of this study is to determine the most effective architectural and geometric parameters by optimizing strut angle and macropore size within the scaffolds to further improve fusion rates of the HBC. METHODS Sixty female Sprague-Dawley rats underwent L4-L5 PLF with placement of one of five HBC scaffold iterations (N=12). Each scaffold consisted of 30vol.% PLGA—a biodegradable polymer—and 70vol.% HA and DBM particles at a proportion of 3:1 HA:DBM. One treatment group received a “solid” scaffold containing no separated struts (no macropores), whereas the remaining four treatment groups received scaffolds with varying combinations of strut angle and macropore size: 90°/1000µm, 45°/1000µm, 90°/500µm, or 45°/500µm. Fusion was evaluated 12 weeks postoperatively using plain radiographs, manual palpation, and microCT imaging. Fusion scoring was assessed by three blinded investigators using an established scoring system: 0=no fusion (motion between segments), 1=unilateral fusion (motion unilaterally), and 2=bilateral fusion (no motion between segments). Spines that received an average score ≥ 1.0 were considered successfully fused. RESULTS The 45°/1000µm scaffold resulted in the highest average fusion score (1.61). Both 90°/500µm and 90°/1000µm scaffolds scored 1.42, whereas the 45°/500µm and solid scaffold resulted in average fusion scores of 0.917 and 0.333, respectively. Animals treated with the 45°/1000µm and 90°/500 µm had fusion rates of 100%. The 90°/1000, 45°/500µm, and solid scaffolds achieved fusion rates of 83.3% and 58.3% and 16.7% respectively. CONCLUSIONS The HBC scaffold represents a novel biomaterial whose success in a clinically relevant rat model suggests that the HBC has significant potential as a BGS. Further studies will compare the efficacy of the HBC composites with that of rhBMP-2 delivered on an absorbable collagen sponge (INFUSE) in promoting spinal fusion.

In vitro and in vivo evaluation of xenogeneic bone putty with the carrier of hydrogel derived from demineralized bone matrix.

The demineralized bone matrix (DBM) putty is a traditional bone graft utilized to facilitate the repair and reconstruction of bone. Recent studies indicated the DBM putties with the various carriers were different in bone repairing ability. In order to prepare a kind of DBM putty with a good biocompatibility and bioactivity, the DBM gel was processed from the DBM and the feasibility as a carrier for the DBM putty was evaluated. After the bovine DBM gel was prepared, the BMPs content as well as the ability to promote osteogenic differentiation of MC3T3-E1 cells in vitro were investigated. Then the DBM putty was prepared and filled into the rat calvarial defect model to evaluate the bone repairing ability by micro-CT and histology. The result showed there was 2.953 ± 0.054ng BMP contained in per gram of the DBM gel. And the ALP production of MC3T3-E1 cells in the DBM gels group increased with prolonged culturing, the mineralized nodules formed in MC3T3-E1 cells on 14th day after co-culture. The putty prepared by DBM gel was easy to handle without loss of DBM particles at room temperature. In the rat calvarial bone defect experiment, histological observation showed more mature bone formed in the DBM putty group than that in the type I collagen group at 12weeks, which indicated the bone putty prepared by DBM gel exhibited a better bone repair capability.

When size matters: differences in demineralized bone matrix particles affect collagen structure, mesenchymal stem cell behavior, and osteogenic potential.

Demineralized bone matrix (DBM) is a natural, collagen-based, osteoinductive biomaterial. Nevertheless, there are conflicting reports on the efficacy of this product. The purpose of this study was to evaluate whether DBM collagen structure is affected by particle size and can influence DBM cytocompatibility and osteoinductivity. Sheep cortical bone was ground and particles were divided in three fractions with different sizes, defined as large (L, 1-2 mm), medium (M, 0.5-1 mm), and small (S, <0.5 mm). After demineralization, the chemical-physical analysis clearly showed a particle size-dependent alteration in collagen structure, with DBM-M being altered but not as much as DBM-S. DBM-M displayed a preferable trend in almost all biological characteristics tested, although all DBM particles revealed an optimal cytocompatibility. Subcutaneous implantation of DBM particles into immunocompromised mice resulted in bone induction only for DBM-M. When sheep MSC were seeded onto particles before implantation, all DBM particles were able to induce new bone formation with the best incidence for DBM-M and DBM-S. In conclusion, the collagen alteration in DBM-M is likely the best condition to promote bone induction in vivo. Furthermore, the choice of 0.5-1 mm particles may enable to obtain more efficient and consistent results among different research groups in bone tissue-engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1019-1033, 2017.

Osteoinductive PolyHIPE Foams as Injectable Bone Grafts.

We have recently fabricated biodegradable polyHIPEs as injectable bone grafts and characterized the mechanical properties, pore architecture, and cure rates. In this study, calcium phosphate nanoparticles and demineralized bone matrix (DBM) particles were incorporated into injectable polyHIPE foams to promote osteoblastic differentiation of mesenchymal stem cells (MSCs). Upon incorporation of each type of particle, stable monoliths were formed with compressive properties comparable to control polyHIPEs. Pore size quantification indicated a negligible effect of all particles on emulsion stability and resulting pore architecture. Alizarin red calcium staining illustrated the incorporation of calcium phosphate particles at the pore surface, while picrosirius red collagen staining illustrated collagen-rich DBM particles within the monoliths. Osteoinductive particles had a negligible effect on the compressive modulus (∼30 MPa), which remained comparable to human cancellous bone values. All polyHIPE compositions promoted human MSC viability (∼90%) through 2 weeks. Furthermore, gene expression analysis indicated the ability of all polyHIPE compositions to promote osteogenic differentiation through the upregulation of bone-specific markers compared to a time zero control. These findings illustrate the potential for these osteoinductive polyHIPEs to promote osteogenesis and validate future in vivo evaluation. Overall, this work demonstrates the ability to incorporate a range of bioactive components into propylene fumarate dimethacrylate-based injectable polyHIPEs to increase cellular interactions and direct specific behavior without compromising scaffold architecture and resulting properties for various tissue engineering applications.

Joint use of developed collagen-containing complexes and cell cultures in creating new tissue equivalents

The purpose of the study is to assess the possibility of applying the integrated module as the basis of a celltissue equivalent for treatment of wounds of skin and soft tissues. In the frame of the set task the following problems were being solved: research of the spatial structure and architectonics of the surface of the developed base collagen-containing materials and their biocompatibility with cell cultures. Materials and methods. The study of a material which is a two-layer complex film, consisting of collagen and polysaccharide components was carried out. The collagen was separated from the dermis and was then impregnated with particulate demineralized bone matrix (DCM) according to the original methodology. For the purposes of the study the dehydrated material was created in the form of a film. Electron microscopic examination of surfaces was performed on scanning electron microscope JEOL JSM-IT300LV in high vacuum and at low values of probe current (< 0,1 nА). Studies to assess the viability of the cells cultivated on films of collagen material (tested for cytotoxicity and the adhesive capacity) were performed in vitro using strains of diploid human fibroblasts 4–6 passage. The culture condition was visually assessed using an inverted Leica microscope DM IL (Carl Zeiss, Austria), equipped with a computerizes program of control of culture growth (Leica IM 1000). Results. The data obtained in the study of the surface structure of the developed complex module showed that it seems to be promising as a basic component of the cellular-tissue system with its large number of structural formations for fixation of the cells and a well-organized barrier layer capable of vapor - permeability. Experiments in vitro confirmed the absence of toxicity of the material being studied in relation to the culture of dermal human fibroblasts, suggesting the possibility of creation on its basis of cell-tissue complex and further experimental studies in vivo. Conclusion. Thus it was experimentally confirmed that the physical characteristics of the developed integrated module satisfy the requirements for the materials for cultivation of cells. The absence of cytotoxicity on the model of a culture of dermal human fibroblasts allows to make a conclusion about a possibility of its use as the basis of cell-tissue equivalent. Preliminary results indicate the advisability of further experiments in vivo aimed at improving complex collagen-containing materials, the development of different ways of their application and clinical evaluation of the effectiveness in the treatment of wounds of various genesis.

Synthetic biodegradable hydrogel delivery of demineralized bone matrix for bone augmentation in a rat model

There exists a strong clinical need for a more capable and robust method to achieve bone augmentation, and a system with fine-tuned delivery of demineralized bone matrix (DBM) has the potential to meet that need. As such, the objective of the present study was to investigate a synthetic biodegradable hydrogel for the delivery of DBM for bone augmentation in a rat model. Oligo(poly(ethylene glycol) fumarate) (OPF) constructs were designed and fabricated by varying the content of rat-derived DBM particles (either 1:3, 1:1 or 3:1 DBM:OPF weight ratio on a dry basis) and using two DBM particle size ranges (50–150 or 150–250μm). The physical properties of the constructs and the bioactivity of the DBM were evaluated. Selected formulations (1:1 and 3:1 with 50–150μm DBM) were evaluated in vivo compared to an empty control to investigate the effect of DBM dose and construct properties on bone augmentation. Overall, 3:1 constructs with higher DBM content achieved the greatest volume of bone augmentation, exceeding 1:1 constructs and empty implants by 3- and 5-fold, respectively. As such, we have established that a synthetic, biodegradable hydrogel can function as a carrier for DBM, and that the volume of bone augmentation achieved by the constructs correlates directly to the DBM dose.

Hydrogels derived from demineralized and decellularized bone extracellular matrix

The extracellular matrix (ECM) of mammalian tissues has been isolated, decellularized and utilized as a scaffold to facilitate the repair and reconstruction of numerous tissues. Recent studies have suggested that superior function and complex tissue formation occurred when ECM scaffolds were derived from site-specific homologous tissues compared with heterologous tissues. The objectives of the present study were to apply a stringent decellularization process to demineralized bone matrix (DBM), prepared from bovine bone, and to characterize the structure and composition of the resulting ECM materials and DBM itself. Additionally, we sought to produce a soluble form of DBM and ECM which could be induced to form a hydrogel. Current clinical delivery of DBM particles for treatment of bone defects requires incorporation of the particles within a carrier liquid. Differences in osteogenic activity, inflammation and nephrotoxicity have been reported with various carrier liquids. The use of hydrogel forms of DBM or ECM may reduce the need for carrier liquids. DBM and ECM hydrogels exhibited sigmoidal gelation kinetics consistent with a nucleation and growth mechanism, with ECM hydrogels characterized by lower storage moduli than the DBM hydrogels. Enhanced proliferation of mouse primary calvarial cells was achieved on ECM hydrogels, compared with collagen type I and DBM hydrogels. These results show that DBM and ECM hydrogels have distinct structural, mechanical and biological properties and have the potential for clinical delivery without the need for carrier liquids.