Comparative Osteogenesis of Three Clinical Bone Graft Materials: An In Vivo Study.

PURPOSE: To investigate bone regeneration among three different bone graft materials in a rat calvarum model. MATERIALS AND METHODS: A total of 24 rats had two 5-mm defects placed per calvarial. Rats were divided into four groups: bovine xenograft (XG), demineralized bone matrix (DBM), mineralized bone graft (MBG), and collagen membrane control (CC). Within each group, samples were collected at two time points: 4 weeks (T4) and 8 weeks (T8). Bone regeneration was assessed by microcomputed tomography (micro-CT) imaging and was analyzed using MATLAB software. Additionally, the fixed samples were subsequently demineralized for immunohistochemistry and histomorphometry. Slides were mounted and stained with hematoxylin and eosin (H&E) stain as well as bone morphogenetic protein 2 (BMP-2) and runt-related transcription factor 2 (RUNX2) markers. The numbers of positive cells/area were calculated for each group and analyzed. RESULTS: At 4 weeks, DBM showed low mineral density (7.7%) compared to the control (25.2%), but increased dramatically at 8 weeks (DBM, T8 = 27.6%; CC, T8 = 27.2%). Xenograft material showed an increase in mineral desnity between T4 and T8 (XG, T4 = 25.0%; XG, T8 = 32.3%). MBG remained consistent over the 8-week trial period (MBG, T4 = 30.4%; MBG, T8 = 30.4%). BMP-2 expression was present in cells adherent to all graft materials. RUNX2 expression was also observed in cells adherent to all graft materials, indicating that during the 4- to 8-week healing period, all materials supported osteogenesis. CONCLUSIONS: Compared to other materials, the DBM had high osteoinductive properties during the 4- to 8-week time period based on increased mineral content. All materials were associated with immunohistologic evidence of osteogenesis in the rat calvarial defect model.

Effects of demineralization mode and particle size of allogeneic bone powder on its physical and chemical properties.

At present, the commonly used allogeneic bone powder in the clinic can be divided into nondemineralized bone matrix and demineralized bone matrix (DBM). Commonly used demineralizers include acids and ethylene diamine tetraacetic acid (EDTA). There may be some diversities between them. Also, the size of the bone particle can affects its cell compatibility and osteogenic ability. We produced different particle sizes i.e., < 75, 75-100, 100-315, 315-450, 450-650, and 650-1000 µm, and treated in three ways (nondemineralized, demineralized by EDTA, and demineralized by HCl). Scanning electron microscopy showed that the surface of the samples in each group was relatively smooth without obvious differences. The results of specific surface area and porosity analysis showed that they were significantly higher in demineralized bone powder than in nondemineralized bone powder, however, there was no significant difference between the two decalcification methods. The content of hydroxyproline in nondemineralized bone powder and EDTA-demineralized bone powder had no statistical difference, while HCl-demineralization had statistical significance compared with the former two, and the content increased with the decrease of particle size. The protein and BMP-2 extracted from HCl demineralized bone powder were significantly higher than that from nondemineralized bone powder and EDTA demineralized bone powder, and there were differences among different particle sizes. These results suggested the importance of demineralization mode and particle size of the allogenic bone powder and provided guidance for the choice of the most appropriate particle size and demineralization mode to be used in tissue bioengineering.

Characterization of an advanced viable bone allograft with preserved native bone-forming cells.

Bone grafts are widely used to successfully restore structure and function to patients with a broad range of musculoskeletal ailments and bone defects. Autogenous bone grafts are historically preferred because they theoretically contain the three essential components of bone healing (ie, osteoconductivity, osteoinductivity, and osteogenicity), but they have inherent limitations. Allograft bone derived from deceased human donors is one alternative that is also capable of providing both an osteoconductive scaffold and osteoinductive potential but, until recently, lacked the osteogenic component of bone healing. Relatively new, cellular bone allografts (CBAs) were designed to address this need by preserving viable cells. Although most commercially-available CBAs feature mesenchymal stem cells (MSCs), osteogenic differentiation is time-consuming and complex. A more advanced graft, a viable bone allograft (VBA), was thus developed to preserve lineage-committed bone-forming cells, which may be more suitable than MSCs to promote bone fusion. The purpose of this paper was to present the results of preclinical research characterizing VBA. Through a comprehensive series of in vitro and in vivo assays, the present results demonstrate that VBA in its final form is capable of providing all three essential bone remodeling properties and contains viable lineage-committed bone-forming cells, which do not elicit an immune response. The results are discussed in the context of clinical evidence published to date that further supports VBA as a potential alternative to autograft without the associated drawbacks.

Evaluation of the Growth and Differentiation of Human Fetal Osteoblasts (hFOB) Cells on Demineralized Bone Matrix (DBM).

Cells with osteogenic potential are believed to be an ideal source for bone tissue bioengineering. Large bone defects require temporary substitution of the damaged parts. In this respect, the transplantation of bone cells cultured on osteogenic substrates has been investigated. To use the natural bone matrix, one approach is the so-called demineralized bone matrix (DBM). In this study, we evaluated the interaction of human fetal osteoblasts (hFOB 1.19 cells, a human fetal osteoblastic cell line) with DBM fragments. No additional bone differentiation inducer was used other than the DBM itself. The samples were processed, had adhesion pattern evaluated and analyzed by light microscopy (cytochemical and immunocytochemical analysis) and electron microscopy (scanning and transmission). The adhesion pattern of hFOB cells on DBM was similar to what was observed on the cell culture plate. Morphological analysis showed that the hFOB cells had emitted filopodia and cellular projections on both controls and DBM. On DBM, the adhered cells emitted prolongations and migrated into the matrix. The monolayer growth pattern was observed as well as the accumulation of filamentous and reticulate extracellular materials when hFOB cells were cultured on the DBM surface. EDS analysis revealed the deposition of calcium on DBM. Immunocytochemical data showed that the hFOB cells were able to secrete extracellular matrix molecules such as fibronectin and laminin on DBM. Our data indicate that DBM successfully stimulates the osteoblastic phenotype of osteoblast-like cells and corroborate with the fact that DBM is a considerable natural matrix that promotes fractured-bone healing.

Co-delivery of simvastatin and demineralized bone matrix hierarchically from nanosheet-based supramolecular hydrogels for osteogenesis.

Supramolecular hydrogels are widely used as 3D scaffolds and delivery platforms in tissue engineering applications. However, hydrophobic therapeutic agents exhibit weak compatibility in hydrogel scaffolds along with aggregation and precipitation. Herein, simvastatin drugs used as BMP-2 stimulators are encapsulated into the layer space of LAPONITE® via electrostatic interactions and ion exchange efficiently, and supramolecular hydrogels could be fabricated with a self-healing, injectable and sustained drug release nature. Hydrogels encapsulated with 10 µg mL-1 simvastatin drug show good osteogenic differentiation in vitro. Moreover, the loading of demineralized bone matrix particles could enhance the capacity for osteogenesis via improving the expression of BMP-2 synergistically. The integrated hydrogels could be implanted into cranial defect sites for bone regeneration in vivo. This work provides the first demonstration of molecular and supramolecular engineering of hydrogels to load osteoinductive agents hierarchically for bone regeneration, contributing to the development of a brand-new strategy for dealing with compatibility between scaffolds and osteogenic agents.

In Vivo Biological Behavior of Polymer Scaffolds of Natural Origin in the Bone Repair Process.

Autologous bone grafts, used mainly in extensive bone loss, are considered the gold standard treatment in regenerative medicine, but still have limitations mainly in relation to the amount of bone available, donor area, morbidity and creation of additional surgical area. This fact encourages tissue engineering in relation to the need to develop new biomaterials, from sources other than the individual himself. Therefore, the present study aimed to investigate the effects of an elastin and collagen matrix on the bone repair process in critical size defects in rat calvaria. The animals (Wistar rats, n = 30) were submitted to a surgical procedure to create the bone defect and were divided into three groups: Control Group (CG, n = 10), defects filled with blood clot; E24/37 Group (E24/37, n = 10), defects filled with bovine elastin matrix hydrolyzed for 24 h at 37 °C and C24/25 Group (C24/25, n = 10), defects filled with porcine collagen matrix hydrolyzed for 24 h at 25 °C. Macroscopic and radiographic analyses demonstrated the absence of inflammatory signs and infection. Microtomographical 2D and 3D images showed centripetal bone growth and restricted margins of the bone defect. Histologically, the images confirmed the pattern of bone deposition at the margins of the remaining bone and without complete closure by bone tissue. In the morphometric analysis, the groups E24/37 and C24/25 (13.68 ± 1.44; 53.20 ± 4.47, respectively) showed statistically significant differences in relation to the CG (5.86 ± 2.87). It was concluded that the matrices used as scaffolds are biocompatible and increase the formation of new bone in a critical size defect, with greater formation in the polymer derived from the intestinal serous layer of porcine origin (C24/25).

Biohybrid Bovine Bone Matrix for Controlled Release of Mesenchymal Stem/Stromal Cell Lyosecretome: A Device for Bone Regeneration.

SmartBone® (SB) is a biohybrid bone substitute advantageously proposed as a class III medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic, and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed. SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell (MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs (38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other hand, the process was still present, but tissue formation was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless, SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-ß.

Biomimetic 3D-Bone Tissue Model.

Various approaches have been evaluated for developing three-dimensional (3D) scaffolds for modeling or engineering of the bone tissue. However, most of such attempts have come up short in mimicking the natural bone tissue extracellular matrix (ECM) microenvironment, especially its natural bioactive content. Here we describe the methodology for the preparation of a natural ECM-based multichannel construct as a biomimetic 3D bone tissue model. We elucidate the construction of the composite scaffold incorporating decellularized small intestinal submucosa ECM, synthetic hydroxyapatite and poly(ε-caprolactone), and the mechanical stimulation of the cell-seeded construct under bioreactor culture.

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

BACKGROUND Our objective was to explore a synthetic alginate hydrogel delivery system for the delivery of demineralized bone matrix (DBM) particles for bone graft substitutes. MATERIAL AND METHODS The 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. RESULTS DBM 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. CONCLUSIONS The delivery of DBM particles using a synthetic alginate hydrogel carrier may be a promising approach in bone tissue engineering for bone defects.

Bioartificial injectable cartilage implants from demineralized bone matrix/PVA and related studies in rabbit animal model.

Functional cartilage tissue engineering needs a substantial, easy to handle scaffold with proper mechanical strength to repair defected area in articular cartilage. In this study, we report the development and characterization of demineralized bone matrix (DBM) in with a poly vinyl alcohol (PVA) to have a proper homogenous injectable scaffold. Injectabiliy of the biodegradable scaffolds, degradation rate, swelling ratio compression and tensile mechanical properties, and viability and proliferation of bone marrow mesenchymal stem cells (BM-MSCs) followed by differentiation of them In-vitro and In-vivo seeded within the scaffold were studied. It demonstrated that the PVA 20% could increase significantly (p < 0.05) the biodegradability of DBM after 720 hours.DBM with 20% of PVA scaffold has significantly higher (p < 0.05) compression and tensile mechanical strength and viscosity. SEM images showed a multilayer of cells on DBM scaffold incorporated with PVA 20%.BM-MSCs on scaffolds, DBM+PVA 20% had a significant growth rate (p < 0.0001) compare to 2D and low concentration of PVA after 21 days of culture. Viability of cells was significantly higher (p < 0.05) on DBM+PVA scaffold compare to DBM. DBM+PVA 20% enhanced cell viability (P < 0.05) compare to DBM scaffold. The PVA presence enhanced chondrogenesis differentiation at the cellular and molecular levels, as evidenced by increased COL II (P < 0.05) and SOX2 upregulation of Chondrogensis-specific genes (p < 0.001). Hyline-like cartilage covered the defect which was confirmed by microscopy and histology assessments. Having considered percentages of PVA with a constant amount of DBM, injectability, compressive mechanical properties, homogeneity of the scaffold, and providing sufficient surface area (12.25 cm2/ml) for cell attachment; 0.35 g/ml of DBM in 20% PVA (w/v) has applicable properties within the ranges of studies which can be proposed for the injectable engineered articular cartilage.

Probing short and long-range interactions in native collagen inside the bone matrix by BioSolids CryoProbe.

Solid-state nuclear magnetic resonance is a promising technique to probe bone mineralization and interaction of collagen protein in the native state. However, many of the developments are hampered due to the low sensitivity of the technique. In this article, we report solid-state nuclear magnetic resonance (NMR) experiments using the newly developed BioSolids CryoProbe™ to access its applicability for elucidating the atomic-level structural details of collagen protein in native state inside the bone. We report here approximately a fourfold sensitivity enhancement in the natural abundance 13 C spectrum compared with the room temperature conventional solid-state NMR probe. With the advantage of sensitivity enhancement, we have been able to perform natural abundance 15 N cross-polarization magic angle spinning (CPMAS) and two-dimensional (2D) 1 H-13 C heteronuclear correlation (HETCOR) experiments of native collagen within a reasonable timeframe. Due to high sensitivity, 2D 1 H/13 C HETCOR experiments have helped in detecting several short and long-range interactions of native collagen assembly, thus significantly expanding the scope of the method to such challenging biomaterials.

Human placenta-derived matrix with cancellous autograft and demineralized bone matrix for large segmental long-bone defects in two dogs with septic nonunion.

OBJECTIVE: To report the successful treatment of septic nonunion in two dogs with large segmental defects secondary to long-bone fractures by using a novel human placenta-derived matrix (hPM) as adjunct to fixation. ANIMALS: One 3-kg 9-year-old neutered male Yorkshire terrier with a distal antebrachial fracture and one 6-kg 4-year-old spayed female miniature pinscher with a distal humeral fracture. STUDY DESIGN: Short case series. METHODS: Both dogs presented for septic nonunion after internal fixation of Gustilo type II open diaphyseal fractures from dog bite injuries. During revision, debridement of nonviable bone resulted in segmental defects of 32% and 20% of the bone length for the antebrachial and humeral fractures, respectively. The antebrachial fracture was stabilized with a circular external fixator, and the humeral fracture was stabilized with biaxial bone plating. The fracture sites were not collapsed, and full length was maintained with the fixation. Autogenous cancellous bone graft and canine demineralized bone allograft were packed into the defects, and hPM was injected into the graft sites after closure. RESULTS: Radiographic union was documented at 8 weeks and 6 weeks for the antebrachial and humeral fractures, respectively. Both dogs became fully weight bearing on the affected limbs and returned to full activity. CONCLUSION: Augmenting fixation with grafts and hPM led to a relatively rapid union in both dogs reported here.

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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. CLINICAL RELEVANCE: 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.

Demineralized bone matrix paste formulated with biomimetic PLGA microcarriers for the vancomycin hydrochloride controlled delivery: Release profile, citotoxicity and efficacy against S. aureus.

Infection and resulting bone defects caused by Staphylococcus aureus is one of the major issues in orthopaedic surgeries. Vancomycin hydrochloride (VaH) is largely used to manage these events. Here, a human derived bone paste supplemented with biopolymer microcarriers for VaH sustained delivery to merge osteoinductive and antimicrobial actions is described. In detail, different emulsion formulations were tested to fabricate micro-carriers of poly-lactic-co-glycolic acid (PLGA) and hydroxyapatite (HA) by a proprietary technology (named Supercritical Emulsion Extraction). These carriers (mean size 827 ± 68 µm; loading 47 mgVaH/gPLGA) were assembled with human demineralized bone matrix (DBM) to obtain an antimicrobial bone paste system (250 mg/0.5 cm3 w/v, carrier/DBM). Release profiles in PBS indicated a daily drug average release of about 4 µg/mL over two weeks. This concentration was close to the minimum inhibitory concentration and able to effectively inhibit the S. aureus growth in our experimental sets. Carriers cytotoxicity tests showed absence of adverse effects on cell viability at the concentrations used for paste assembly. This approach points toward the potential of the DBM-carrier-antibiotic system in hampering the bacterial growth with accurately controlled antibiotic release and opens perspectives on functional bone paste with PLGA carriers for the controlled release of bioactive molecules.

A bone matrix-simulating scaffold to alleviate replicative senescence of mesenchymal stem cells during long-term expansion.

Replicative senescence during in vitro augmentation, which is mostly induced by the loss of physiological microenvironment, hinders the application of mesenchymal stem cells (MSCs) in the clinic. Here, we investigated whether MSCs senescence could be prevented by bio-scaffold mimicking the natural tissue matrix. Human umbilical cord mesenchymal stem cells (hUCMSCs) exhibited a senescent phenotype during a long-term passage in the conventional culture dish. To fabricate the bone matrix, a naturally based matrix composed of nano-hydroxyapatite/chitosan/poly lactide-co-glycolide (nHA/CS/PLGA) was produced. Long-term passage resulted in an obvious increase in the expression of senescence markers and a reduction in the expression of master genes involved in tissue regeneration. Functional assay confirmed that nHA/CS/PLGA scaffold preserved the proliferation and differentiation of hUCMSCs even after being passaged 27 times. Moreover, in vivo ectopic bone formation assay revealed that the bone formation of hUCMSCs cultured on the nano-scaffolds for the long term was as robust as the cells in the early passage. In summary, our results demonstrate that nHA/CS/PLGA scaffold effectively preserves the stemness and youth of hUCMSCs in the long-term passage. Taken advantage of its compatibility and bioactivity, nHA/CS/PLGA scaffold is of great potential in large-scale expansion of MSCs for stem cell therapy and tissue engineering.

Photobiomodulation Therapy Associated with Heterologous Fibrin Biopolymer and Bovine Bone Matrix Helps to Reconstruct Long Bones.

Bone defects cause aesthetic and functional changes that affect the social, economic and especially the emotional life of human beings. This complication stimulates the scientific community to investigate strategies aimed at improving bone reconstruction processes using complementary therapies. Photobiomodulation therapy (PBMT) and the use of new biomaterials, including heterologous fibrin biopolymer (HFB), are included in this challenge. The objective of the present study was to evaluate the influence of photobiomodulation therapy on bone tibial reconstruction of rats with biomaterial consisting of lyophilized bovine bone matrix (BM) associated or not with heterologous fibrin biopolymer. Thirty male rats were randomly separated into three groups of 10 animals. In all animals, after the anesthetic procedure, a noncritical tibial defect of 2 mm was performed. The groups received the following treatments: Group 1: BM + PBMT, Group 2: BM + HFB and Group 3: BM + HFB + PBMT. The animals from Groups 1 and 3 were submitted to PBMT in the immediate postoperative period and every 48 h until the day of euthanasia that occurred at 14 and 42 days. Analyses by computed microtomography (µCT) and histomorphometry showed statistical difference in the percentage of bone formation between Groups 3 (BM + HB + PBMT) and 2 (BM + HFB) (26.4% ± 1.03% and 20.0% ± 1.87%, respectively) at 14 days and at 42 days (38.2% ± 1.59% and 31.6% ± 1.33%, respectively), and at 42 days there was presence of bone with mature characteristics and organized connective tissue. The µCT demonstrated BM particles filling the defect and the deposition of new bone in the superficial region, especially in the ruptured cortical. It was concluded that the association of PBMT with HFB and BM has the potential to assist in the process of reconstructing bone defects in the tibia of rats.

Improved physical and osteoinductive properties of demineralized bone matrix by gelatin methacryloyl formulation.

The demineralized bone matrix (DBM) is the most widely used bone allograft, which is obtained by removing the mineral component of bone, leading to exposure of the proteins responsible for osteoinduction. For clinical use, DBM shall be formulated with a carrier that provides consistency and improves its osteoinduction. In this study, three DBM formulations with glycerol (Gly), hyaluronic acid (HA), and gelatin methacryloyl (GelMA) were evaluated measuring their physicochemical properties (microstructure, compressive strength, and serum cohesivity) and their osteoinductive capacity both in vitro using C2C12 cells and umbilical cord human mesenchymal stem cells and in vivo in an ectopic bone formation model in athymic mice. To assess the effectiveness of DBM in vitro in inducing the differentiation into osteoblasts, alkaline phosphatase (ALP) activity was assessed in combination with a cytotoxicity assay. In vivo, new bone formation was assessed by histological and radiological analysis. In the compression and in the serum cohesive assays, the GelMA DBM formulation showed its superiority over the other formulations. In addition, GelMA showed a more compact structure analysed by scanning electron microscopy. Higher cell toxicity was observed on Gly formulations in vitro, whereas GelMa and HA showed very low toxicity. All formulations significantly improved ALP activity compared with control. In the in vivo studies, GelMA showed the greatest osteoinductive potential with a higher percentage of new bone and bone marrow formation. Our results suggest GelMA is useful as a carrier for DBM designed to promote the formation of the new bone.

In vitro and in vivo assessment of CaP materials for bone regenerative therapy. The role of multinucleated giant cells/osteoclasts in bone regeneration.

In this work, bone formation/remodeling/maturation was correlated with the presence of multinucleated giant cells (MGCs)/osteoclasts (tartrate-resistant acid phosphatase [TRAP]-positive cells) on the surface of beta-tricalcium phosphate (ß-TCP), sintered deproteinized bovine bone (sDBB), and carbonated deproteinized bovine bone (cDBB) using a maxillary sinus augmentation (MSA) in a New Zealand rabbit model. Microtomographic, histomorphometric, and immunolabeling for TRAP-cells analyses were made at 15, 30, and 60 days after surgery. In all treatments, a faster bone formation/remodeling/maturation and TRAP-positive cells activity occurred in the osteotomy region of the MSA than in the middle and submucosa regions. In the ß-TCP, the granules were rapidly reabsorbed by TRAP-positive cells and replaced by bone tissue. ß-TCP enabled quick bone regeneration/remodeling and full bone and marrow restoration until 60 days, but with a significant reduction in MSA volume. In cDBB and sDBB, the quantity of TRAP-positive cells was smaller than in ß-TCP, and these cells were associated with granule surface preparation for osteoblast-mediated bone formation. After 30 days, more than 80% of granule surfaces were surrounded and integrated by bone tissue without signs of degradation, preserving the MSA volume. Overall, the materials tested in a standardized preclinical model led to different bone formation/remodeling/maturation within the same repair process influenced by different microenvironments and MGCs/osteoclasts. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:282-297, 2020.

Biphasic calcium phosphate and polymer-coated bovine bone matrix for sinus grafting in an animal model.

Autogenous bone grafting requires a donor site and may lose substantial volume during remodeling. Several bone replacement materials (BRMs) are under development to overcome these limitations, especially for indications for minimally intervention surgeries. The objective of our study was to assess the potential of an equine collagen cone reinforced with biphasic calcium phosphate (CC-BCP) particles and deproteinized bovine bone matrix (BBM) coated with polylactic acid, and poly-ε-caprolactone copolymer (BBM-PCC) and then to compare the outcomes with a deproteinized BBM and an equine CC without a filler in a sheep sinus grafting model in the Elleven female sheep were selected. Two experimental sites on each side of the animals were prepared using an extraoral approach for maxillary sinus wall. The four treatments were performed in each animal through a standardized 10-mm access window. While the BBM access was covered with a collagen membrane, all other sites were closed with an equine collagen membrane. All animals were euthanized after 16 weeks. New bone (NB), residual graft particles, and connective tissue were measured in undemineralized resin-embedded sections. As a result, one sheep did not survive the surgery. All sites in the remaining 10 sheep healed uneventfully. All CC and BBM-PCC grafts resorbed and failed to augment the sinuses. BBM and CC-BCP, in contrast, showed some histologic evidence of NB and surgical site augmentation. The NB fraction in the latter two groups accounted for 10 ± 9 and 4 ± 5%, respectively (p > 0.05). In conclusion, BBM-PCC and collagen cone performed poorly for sinus floor augmentation, while deproteinised BBM and reinforced collagen cone demonstrated comparable outcomes.