Demineralized Bone Matrix Scaffolds Research Articles

Chondrogenic Potential of PMSCs Cultured on Chondroitin Sulfate/Gelatin -Modified DBM Scaffold

Introduction: Osteoarthritis is one of the most common orthopedic diseases that gradually causes wear and damage to the articular Subchondral bone due to the destruction of articular cartilage. One of the basic challenges in cartilage tissue engineering is the choice of scaffold. In the design of the cartilage scaffold, it is useful to consider parameters such as porosity, water absorption, high mechanical resistance, biocompatibility, and biodegradability. Therefore, in this study, Demineralized Bone Matrix (DBM) which inherently has these characteristics to some extent, was chosen as the basic scaffold. Methods: The Gelatin/demineralized bone matrix (G/DBM) and the Chondroitin sulfate- Gelatin/demineralized bone matrix (GCS/DBM) scaffolds were prepared, respectively by incorporating gelatin or Chondroitin sulfate/gelatin solution inside DBM pores, freeze-drying and crosslinking with EDC/NHS. The physicochemical, biological characteristics, and chondrogenic potential of scaffolds were studied. Results: According to the SEM results, the size of the DBM pores in the G/DBM and GCS/DBM scaffolds decreased (from almost, 100-1500 µm to less than 200 µm) which reduced cell escape compared to the DBM scaffold. Also, crosslinking the scaffolds has greatly increased their compressive E-modulus (more than 8 times). The cytocompatibility and non-toxicity of all scaffolds were confirmed by acridine orange/ethidium bromide (AO/EB) staining. The evaluation results of chondrogenic differentiation of placenta-derived mesenchymal stem cells (PMSCs) on modified scaffolds, using the Real-time PCR method, showed that the presence of CS in the GCS/DBM scaffold improved the expression of chondrogenesis markers such as Aggrecan (AGC) (~4 times) and collagen 2 (COL-2) (~ 2.2 times) compared to the DBM scaffold. Also, Alcian blue staining and immunohistochemical analyses of the scaffolds, respectively, showed a denser and more coherent synthesis of GAGs and COL-2 protein on the GCS/DBM scaffold than on the G/DBM and DBM. Conclusion: According to the obtained results, it seems that the GCS/DBM scaffold can be a suitable scaffold in cartilage tissue engineering.

MicroRNA-26 and Related Osteogenic Target Genes Could Play Pivotal Roles in Photobiomodulation and Adipose-Derived Stem Cells-Based Healing of Critical Size Foot Defects in the Rat Model.

Objective: In this study, we aimed to explore the role of MicroRNA-26 in photobiomodulation (PBM)- and adipose-derived stem cell (ADS)-based healing of critical-sized foot fractures in a rat model. Background: PBM and ADS treatments are relatively invasive methods for treating bone defects. Specific and oriented cellular and molecular functions can be induced by applying an appropriate type of PBM and ADS treatment. Methods: A critical size foot defect (CSFD) is induced in femoral bones of 24 rats. Then, a human demineralized bone matrix scaffold (hDBMS) was engrafted into all CSFDs. The rats were randomly allocated into four groups (n = 6): (1) control (hDBMS); (2) hDBMS+human ADSs (hADSs), hADSs engrafted into CSFDs; (3) hDBMS+PBM, CSFD exposed to PBM (810 nm wavelength, 1.2 J/cm2 energy density); and (4) hDBMS+(hADSs+PBM), hADSs implanted into the CSFD and then exposed to PBM. At 42 days after CSFD induction, the rats were killed, and the left CSFD was removed for mechanical compression tests and the right CSFD was removed for molecular and histological studies. Results: The results indicate that miRNA-26a, BMP, SMAD, RUNX, and OSTREX had higher expression in the treated groups than in the control group. Further, the biomechanical and histological properties of CSFDs in treated groups were improved compared with the control group. Correlation tests revealed a positive relationship between microRNA and improved biomechanical and cellular parameters of CSFDs in the rat model. Conclusions: We concluded that the MicroRNA-26 signaling pathway probably plays a significant role in the hADS-, PBM-, and hADS+PBM-based healing of CSFDs in rats. Clinical Trial Registration number: IR.SBMU.MSP.REC.1398.980.

Fibrocartilage extracellular matrix augmented demineralized bone matrix graft repairs tendon-to-bone interface in a rabbit tendon reconstruction model

Current tendon/ligament reconstructions integrate via scar tissue rather than proper bone-tendon interface regeneration, which affects graft longevity, changes in bone tunnel size, and functional outcomes. The purpose of this study was to develop a functional demineralized bone matrix (DBM) + fibrocartilage extracellular matrix (FCECM) composite scaffold, characterize its physicochemical properties, and evaluate its efficacy in repairing tendon-bone interface in a rabbit tendon reconstruction model. Solubilized FCECM was loaded and crosslinked on to DBM scaffolds via gamma-irradiation to create DBM + FCECM scaffolds. The resulting scaffold showed interconnected pores coated with FCECM and protein cargo similar to FCECM. The addition of FCECM modified the physicochemical properties of the DBM scaffold, including microstructure, biochemical composition, mechanical strength, thermodynamic properties, and degradation period. The DBM + FCECM scaffold was biocompatible for mesenchymal stem cells (MSCs) and resulted in elevation of fibrochondrogenic gene markers compared to DBM scaffolds in vitro. In vivo implantation of DBM + FCECM scaffold resulted in neofibrocartilage formation, better pullout strength, and less bone tunnel widening compared to DBM only group in a rabbit tendon reconstruction model. In conclusion, the FCECM augmented DBM scaffold repairs the tendon-bone interface with osseous-fibrocartilage tissue, which may be utilized to improve current tendon reconstruction surgeries.

Human Hematopoietic Stem Cells Co-cultured in 3D with Stromal Support to Optimize Lentiviral Vector-mediated Gene Transduction.

HSC transplantation (HSCT) has emerged as a promising treatment option for hematological and immunological disorders. Unfortunately, many viral vectors are inefficient at transduction, limiting the number of cells available for gene therapy in cord blood HSC transplantation. Combining ex vivo expansion and genetic manipulation of cord blood cells is a potential gene therapy approach. We present a 3D co-culture method using a demineralized bone matrix scaffold to optimize lentiviral vector-mediated gene transduction. pLenti-III-miR-GFP-has-miR-124 was transduced into cord blood HSCs. Transduced CD34 + cells co-cultured on the stromal layer for 72h under cytokine-free conditions. We performed flow cytometry, colony assays, real-time polymerase chain reaction, and SEM morphological analysis. Seventy-two hours after transduction, when pLentiIII-miR-GFP-has-miR-124 and control vector-transduced expanded cord blood HSCs were compared to non-transduced expanded cord blood HSCs, the findings revealed 15 ± 3.04 and 55 ± 3.05-fold increases in miR-124 mRNA expression, respectively. Compared to a control culture on the same day, the expansion of CD34+, CD38-HSCs in 3D culture increased 544 ± 31.09 fold. This result demonstrated that the 3D-culture system could emerge as a novel approach to overcoming the current limitations of cord blood HSC transduction. In the future, this research could be applied in a therapeutic setting.

A cell-free difunctional demineralized bone matrix scaffold enhances the recruitment and osteogenesis of mesenchymal stem cells by promoting inflammation resolution.

The dialogue between host macrophages (Mφs) and endogenous mesenchymal stem cells (MSCs) promotes M2 Mφs polarization to resolve early-stage inflammation, thereby effectively guiding in situ bone regeneration. Once inflammation is unresolved/incontrollable, it will induce the impediment of MSCs homing at bone defect site, implying the seasonable resolution of inflammation in balancing bone homeostasis. Repeatedly, evidence elucidated that specialized pro-resolving mediators (SPMs) could conduce to proper resolve inflammation and promote the repairing of bone defect. A difunctional demineralized bone matrix (DBM) scaffold co-modified by maresin 1 (MaR1) and aptamer 19S (Apt19S) was fabricated to facilitate the osteogenesis of MSCs. To confirm the osteogenesis and immunomodulatory role of the difunctional DBM scaffold, the proliferation, recruitment, and osteogenic differentiation of MSCs and the Mφs M2 subtype polarization were evaluated in vitro. Then, the DBM scaffolds were implanted into mice model with critical size calvarial defect to evaluate bone repair efficiency. Finally, the specific resolution mechanism in Mφs cultured on the difunctional DBM scaffold was further in-depth investigated. This difunctional DBM scaffold exhibited an enhanced function on the recruitment, proliferation, migration, osteogenesis of MSCs and the resolution of inflammation, finally improved bone-scaffold integration. At the same time, MaR1 modified on the difunctional DBM scaffold increased the biosynthesis of 12-lipoxygenase (12-LOX) and 12S-hydroxy-eicosatetraenoic acid (12S-HETE), and also directly stimulated lipid droplets (LDs) biogenesis in Mφs, which suggested that MaR1 regulated Mφ lipid metabolism at bone repair site. Findings based on this synergy strategy demonstrated that Mφ lipid metabolism was essential in bone homeostasis, which might provide a theoretical direction for the treatment-associated application of MaR1 in inflammatory bone disease.

In vitro dynamic perfusion of prevascularized OECs-DBMs (outgrowth endothelial progenitor cell - demineralized bone matrix) complex fused to recipient vessels in an internal inosculation manner

ABSTRACT The current research on seed cells and scaffold materials of bone tissue engineering has achieved milestones. Nevertheless, necrosis of seed cells in center of bone scaffold is a bottleneck in tissue engineering. Therefore, this study aimed to investigate the in vivo inosculation mechanism of recipient microvasculature and prevascularized outgrowth endothelial progenitor cells (OECs)-demineralized bone matrix (DBM) complex. A dorsal skinfold window-chamber model with tail vein injection of Texas red-dextran was established to confirm the optimal observation time of microvessels. OECs-DBM complex under static and dynamic perfusion culture was implanted into the model to analyze vascularization. OECs-DBM complex was harvested on 12th day for HE staining and fluorescent imaging. The model was successfully constructed, and the most appropriate time to observe microvessels was 15 min after injection. The ingrowth of recipient microvessels arcoss the border of OECs-DBM complex increased with time in both groups, and more microvessels across the border were observed in dynamic perfusion group on 3rd, 5th, 7th day. Fluorescent integrated density of border in dynamic perfusion group was higher at all-time points, and the difference was more significant in central area. Fluorescent imaging of OECs-DBM complex exhibited that no enhanced green fluorescent protein-positive cells were found beyond the verge of DBM scaffold in both groups. In vitro prevascularization by dynamic perfusion culture can increase and accelerate the blood perfusion of OECs-DBM complex obtained from recipient microvasculature by internal inosculation. Accordingly, this approach may markedly contribute to the future success of tissue engineering applications in clinical practice.

Photobiomodulation and Stem Cell on Repair of Osteoporotic Bones

Objective: This study examined the use of photobiomodulation (PBM) plus adipose-derived stem cells (ASCs) to enhance the osteogenic properties of demineralized bone matrix (DBM) scaffold in a critical size femoral defect (CSFD) of ovariectomy-induced osteoporotic (OVX) rats. Background: PBM could be used as a unique strategy to enhance the osteogenic potential of DBMs seeded with ASCs. Materials and methods: The OVX rats with a CSFD were divided into six groups: (1) Control (C); (2) DBM scaffold alone (S); (3) S+PBM; (4) S+alendronate; (5) S+ASC; (6) S+PBM+ASC. Stereological analysis, real-time polymerase chain reaction (RT-PCR), and cone-beam computed tomography (CBCT) were performed after euthanization at 4 and 8 weeks postimplantation surgery. Results: In the 8th week, Groups 4 and 6 showed a greatly high new trabecular bone volume than the scaffold group (all, p = 0.009). The CBCT data demonstrated that the CSFD was significantly smaller in the two, three, and six groups relative to the control group (p = 0.01, p = 0.000, and p = 0.000, respectively). RT-PCR revealed that Groups 3 and 6 had higher messenger RNA levels of osteocalcin (OC) and osteoprotegerin (OPG) compared with the control group (p = 0.05). Group 6 had significantly lower expression of receptor activator of nuclear factor-κB ligand (RANKL) compared with the control group (p = 0.02). Conclusions: The combination of DBM plus PBM plus ASC, as well as DBM plus PBM significantly improved the healing of CSFD in OVX rats, and affected the expression of OPG, OC, and RANKL genes.

Combining Platelet Rich Plasma and Concentrated Bone Marrow with a Demineralized Bone Matrix Scaffold Enhances Growth Factor Release Over Time

Combining Platelet Rich Plasma and Concentrated Bone Marrow with a Demineralized Bone Matrix Scaffold Enhances Growth Factor Release Over Time

4-Octyl itaconate modified demineralized bone matrix scaffold improves bone repair by regulating early inflammation

Macrophages are involved in the early inflammation during tissue repair and play a distinctive role in maintaining the stability of the tissue microenvironment. However, it is still very difficult to accurately regulate the phenotypic transformation of macrophages during bone repair. The current study innovatively used 4-octyl itaconate (OI), a cell-permeable derivative of an endogenous anti-inflammatory metabolite itaconate (IA), to regulate the polarization of macrophages and enhance the quality of bone repair. Chitosan (CS) was selected as a bridge in coating OI on demineralized bone matrix (DBM) scaffold to generate three-dimensional (3D) bone integration scaffold (OI/CS/DBM scaffold). Whether this metabolite derivative can exert the same anti-inflammatory effect as the metabolite itself was validated both in vitro and in vivo. In vitro cellular results showed that both OI and OI/CS/DBM scaffold could promote the formation of anti-inflammatory phenotype of macrophages, and the osteogenic differentiation of mesenchymal stem cells (MSCs) was indirectly enhanced by macrophage supernatant medium. In vivo studies of a rat skull defect model further proved that the OI/CS/DBM scaffold could improve the bone integration. In this study, the OI/CS/DBM scaffold was successfully fabricated as a vehicle for delivering OI, which was proved to have the ability to promote bone integration by regulating early inflammation. The present results may provide a novel method to improve bone repair with metabolites derivative to regulate the inflammation.

Effect of demineralized bone matrix modified by laminin α4 chain functional peptide on H-type angiogenesis and osteogenesis to promote bone defect repair

Based on the cell-extracellular matrix adhesion theory in selective cell retention (SCR) technology, demineralized bone matrix (DBM) modified by simplified polypeptide surface was designed to promote both bone regeneration and angiogenesis. Functional peptide of α4 chains of laminin protein (LNα4), cyclic RGDfK (cRGD), and collagen-binding domain (CBD) peptides were selected. CBD-LNα4-cRGD peptide was synthesized in solid phase and modified on DBM to construct DBM/CBD-LNα4-cRGD scaffold (DBM/LN). Firstly, scanning electron microscope and laser scanning confocal microscope were used to examine the characteristics and stability of the modified scaffold. Then, the adhesion, proliferation, and tube formation properties of CBD-LNα4-cRGD peptide on endothelial progenitor cells (EPCs) were detected, respectively. Western blot method was used to verify the molecular mechanism affecting EPCs. Finally, 24 10-week-old male C57 mice were used to establish a 2-mm-length defect of femoral bone model. DBM/LN and DBM scaffolds after SCR treatment were used to repair bone defects in DBM/LN group ( n=12) and DBM group ( n=12), respectively. At 8 weeks after operation, the angiogenesis and bone regeneration ability of DBM/LN scaffolds were evaluated by X-ray film, Micro-CT, angiography, histology, and immunofluorescence staining [CD31, endomucin (Emcn), Ki67]. Material related tests showed that the surface of DBM/LN scaffold was rougher than DBM scaffold, but the pore diameter did not change significantly ( t=0.218, P=0.835). After SCR treatment, DBM/LN scaffold was still stable and effective. Compared with DBM scaffold, DBM/LN scaffold could adhere to more EPCs after the surface modification of CBD-LNα4-cRGD ( P<0.05), and the proliferation rate and tube formation ability increased. Western blot analysis showed that the relative expressions of VEGF, phosphorylated FAK (p-FAK), and phosphorylated ERK1/2 (p-ERK1/2) proteins were higher in DBM/LN than in DBM ( P<0.05). In the femoral bone defect model of mice, it was found that mice implanted with DBM/LN scaffold had stronger angiogenesis and bone regeneration capacity ( P<0.05), and the number of CD31 hiEmcn hi cells increased significantly ( P<0.05). DBM/LN scaffold can promote the adhesion of EPCs. Importantly, it can significantly promote the generation of H-type vessels and realize the effective coupling between angiogenesis and bone regeneration in bone defect repair.

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.

Cartilage tissue engineering using injectable functionalized Demineralized Bone Matrix scaffold with glucosamine in PVA carrier, cultured in microbioreactor prior to study in rabbit model

Using 3D model of injectable scaffolds for cartilage tissue engineering is one of the challenges that should be addressed to avoid invasive surgery for treatment. For this purpose, chondrocytes on Demineralized Bone Matrix (DBM) scaffolds functionalized with glucosamine in 20% polyvinyl alcohol (PVA) as a carrier was applied to the micro-bioreactor in-vitro, then the study was continued on in-vivo stage. Scaffold biocompatibility tests were performed and the mechanical and physicochemical properties were studied showing the fact that DBM was functionalized by Glucosamine, scaffold degradation rate was 53% after 720 h and swelling ratio was 2.5 times after 16 h, injectable scaffold demonstrated better mechanical characteristics (P < 0.05) than other concentrations of PVA. Consequently, in-vitro tests, including live-dead imaging resulting in 99% viability after 14 days (P < 0.001), DAPI staining and scanning electron microscope imaging were performed to determine the number and viability of the cells on the scaffold, showing a cells proliferation property of this group compared with the control after 14 days (P < 0.0001), then relative gene expression was evaluated and protein expression was assessed. The overall chondrogenic gene expression improved (P < 0.05) compared to the control (2D culture). Subsequently, the scaffold were loaded with chondrocytes and injected into the cartilage lesion part After 24 weeks of surgery, MRI and immunocytochemistry were performed. Then all outputs proved that the scaffold plus cell group had a significantly higher topological score (P < 0.0001) than other groups compared to normal cartilage. Finally, studies have shown that transplantation of chondrocytes in DBM, polyvinyl alcohol and glucosamine scaffold through one surgical stage improves cartilage lesion and it can be considered as a breakthrough in tissue engineering.

Demineralized bone matrix scaffold modified with mRNA derived from osteogenically pre-differentiated MSCs improves bone repair

Gene therapy based on mRNA provides a promising approach for bone regeneration. Quick mRNA translation and controlled protein production could be earned by implantation of mRNA-activated scaffold in bone remodeling region. Furthermore, the expression levels of osteogenic-related mRNA in the cytoplasm of osteogenically pre-differentiated mesenchymal stem cells (MSCs) were high and the expression levels were different at different stages of osteogenically differentiated MSCs. This study intended to investigate the effect of osteoinductive-mRNAs (Oi-mRNAs), derived from osteogenically pre-differentiated MSCs at various stages (Day 1 (Oi1-mRNA), Day 3 (Oi3-mRNA), Day 7 (Oi7-mRNA), Day 14 (Oi14-mRNA) and Day 21 (Oi21-mRNA), respectively), on the osteogenic differentiation of MSCs. Further, the Oi-mRNAs combined with cationic polymer polyethylenimine (PEI) were loaded onto demineralized bone matrix (DBM) scaffold (Oi-mRNA/DBM). The results revealed that the Oi1-mRNA, Oi3-mRNA and Oi21-mRNA had no obvious effect on the osteogenic differentiation of MSCs, while the Oi7-mRNA increased the expression of alkaline phosphatase (ALP) and the Oi14-mRNA significantly promoted the expression of osteocalcin (OC) and osteopontin (OPN), and calcium deposition. In addition, the Oi14-mRNA/DBM scaffold could significantly enhance extracellular matrix (ECM) secretion and new collagen formation of MSCs. After being implanted into rat critical-sized cranium defect model, the Oi14-mRNA/DBM scaffold could promote the infiltration of cells and repair of bone defect in vivo. The DBM scaffold loaded with mRNA encoding osteoinductive protein may provide a powerful tool for bone defect repair.

Concentrated Bone Marrow Aspirate and Subacromial Bursa-Derived Cells Demonstrate Similar Cellular Adhesion and Proliferation Potential on Demineralized Bone Matrix Scaffolds for Biologic Augmentation of Rotator Cuff Repair

Concentrated Bone Marrow Aspirate and Subacromial Bursa-Derived Cells Demonstrate Similar Cellular Adhesion and Proliferation Potential on Demineralized Bone Matrix Scaffolds for Biologic Augmentation of Rotator Cuff Repair

Studies on Surface Properties and Cell Adhesion Properties of BSA Modified DBM Scaffold

As a scaffold material for bone tissue engineering, demineralized bone matrix(DBM) has such a limited ability to load cells and growth factors that the surface of the DBM scaffold was modified with bovine serum albumin( BSA) with different concentrations to improve the protein structure and physicochemical properties of the scaffold surface so as to enhance the adhesion of the cells. And the appropriate BSA concentration was explored. Compared with DBM, the scaffold with BSA coating had a smaller pore size and a lower porosity, also, the degradation rate was accelerated and the hydrophilic property was improved. Cells adhesion was observed inside the DBM sca ffold before and after it had been modified, and the BSA modified scaffold had a good cell compatibility. When the concentration of BSA was 20 mg/mL, the adhesion ability of the cells to modified scaffold was significantly increased, and the cell proliferation was facilitated.

Reconstructing Bone with Natural Bone Graft: A Review of In Vivo Studies in Bone Defect Animal Model.

Bone defects caused by fracture, disease or congenital defect remains a medically important problem to be solved. Bone tissue engineering (BTE) is a promising approach by providing scaffolds to guide and support the treatment of bone defects. However, the autologous bone graft has many defects such as limited sources and long surgical procedures. Therefore, xenograft bone graft is considered as one of the best substitutions and has been effectively used in clinical practice. Due to better preserved natural bone structure, suitable mechanical properties, low immunogenicity, good osteoinductivity and osteoconductivity in natural bone graft, decellularized and demineralized bone matrix (DBM) scaffolds were selected and discussed in the present review. In vivo animal models provide a complex physiological environment for understanding and evaluating material properties and provide important reference data for clinical trials. The purpose of this review is to outline the in vivo bone regeneration and remodeling capabilities of decellularized and DBM scaffolds in bone defect models to better evaluate the potential of these two types of scaffolds in BTE. Taking into account the limitations of the state-of-the-art technology, the results of the animal bone defect model also provide important information for future design of natural bone composite scaffolds.

Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure.

Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro. In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5MPa with a frequency of 0.5Hz and 4h per day for 1week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively. Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-β1 growth factor (10ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-β1, respectively. The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.

Restoration of mandibular bone defects with demineralized bone matrix combined with three-dimensional cultured bone marrow-derived mesenchymal stem cells in minipig models.

Mandibular defects, caused by congenital, pathological or iatrogenic insults, can significantly affect patient quality of life. The reconstruction of mandible has recently gained the interest of clinical and tissue engineering researchers. The purpose of this study was to evaluate the effectiveness of three-dimensional (3-D) cultured autologous grafts prepared using bone marrow-derived mesenchymal stem cells (BMSCs) combined with demineralized bone matrix (DBM) scaffolds for the restoration of mandibular defects. Cylindrical defects were created in the mandibular body of minipigs and filled with 3D-cultured BMSCs/DBM autografts, 2D-cultured BMSCs/DBM autografts, DBM material (without cells), or were left unfilled (blank). Using computed tomographic (CT) imaging and histological staining, we found that treatment of mandibular defects using 3-D cultured BMSCs/DBM autografts offered improvements in bone formation over both 2-D cultured autografts and cell-free DBM scaffolds. We found increased osteoid formation in 3D and 2D cultures, with more osteogenic cells present in the 3D constructs. We suggest that 3-D cultured homograft BMSCs combined with DBM scaffolds represents a new strategy for bone reconstruction, with potential future clinical applicability.

Demineralized Bone Scaffolds with Tunable Matrix Stiffness for Efficient Bone Integration.

As a biophysical cue, matrix stiffness can decide the stem cell fate. However, most methods to construct three-dimensional (3D) scaffolds may change the 3D microstructure while altering their mechanical properties. In this study, demineralized bone matrix scaffolds with different compressive modulus (66.06 ± 27.83 MPa (high), 26.90 ± 13.16 MPa (medium), and 0.67 ± 0.14 MPa (low)) were constructed by controlling the decalcification duration (1 h, 12 h, and 5 days), respectively. The pore size and porosity have no significant difference between the scaffolds before and after decalcification. Cell experiments indicated that the low scaffolds could promote the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) in vitro. Rat subcutaneous implantation experiments further demonstrated that the low scaffolds could efficiently improve the cell infiltration, deposition of collagen fibers, and positive osteocalcin and osteopontin expression of endogenous cells as well as angiogenesis. Finally, rabbit femoral condylar defect experiments proved that the low scaffolds could significantly promote the bone repair and integration and stromal cell derived factor-1α/CXC chemokine receptor signal pathway was essential for the stiffness-mediated bone repair. These investigations provided a novel method for fabricating 3D bone grafts with different stiffness, which is also of great significance for studying the effect of stiffness on the biological behavior of MSCs in three dimensions.

Demineralized bone matrix-based microcarrier scaffold favors vascularized large bone regeneration in vivo in a rat model.

Insufficient neo-vascularization of in vivo implanted cell-seeded scaffold remains a major bottleneck for clinical translation of engineered bone formation. Demineralized bone matrix is an ideal bone scaffold for bone engineering due to its structural and biochemical components similar to those of native bone. We hypothesized that the microcarrier form of demineralized bone matrix favors ingrowth of vessels and bone regeneration upon in vivo implantation. In this study, a rat model of femoral vessel pedicle-based bone engineering was employed by filling the demineralized bone matrix scaffolds inside a silicone chamber that surrounded the vessel pedicles, and to compare the efficiency of vascularized bone regeneration between microcarrier demineralized bone matrix and block demineralized bone matrix. The results showed that bone marrow stem cells better adhered to microcarrier demineralized bone matrix and produced more extracellular matrices during in vitro culture. After in vivo implantation, microcarrier demineralized bone matrix seeded with bone marrow stem cells formed relatively more bone tissue than block demineralized bone matrix counterpart at three months upon histological examination. Furthermore, micro-computed tomography three-dimensional reconstruction showed that microcarrier demineralized bone matrix group regenerate significantly better and more bone tissues than block demineralized bone matrix both qualitatively and quantitatively (p < 0.05). Moreover, micro-computed tomography reconstructed angiographic images also demonstrated significantly enhanced tissue vascularization in microcarrier demineralized bone matrix group than in block demineralized bone matrix group both qualitatively and quantitatively (p < 0.05). Anti-CD31 immunohistochemical staining of (micro-) vessels and semi-quantitative analysis also evidenced enhanced vascularization of regenerated bone in microcarrier demineralized bone matrix group than in block demineralized bone matrix group (p < 0.05). In conclusion, the microcarrier form of demineralized bone matrix is an ideal bone regenerative scaffold due to its advantages of osteoinductivity and vascular induction, two essentials for in vivo bone regeneration.