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The repair of critical-sized calvarial defects is a challenging problem for orthopedic surgery. One of the promising strategies of bone bioengineering to enhance the efficacy of large bone defect regeneration is the combined delivery of stem cells with osteoinductive factors within polymer carriers. The purpose of the research was to study the regenerative effects of heparin-conjugated fibrin (HCF) hydrogel containing bone morphogenetic protein 2 (BMP-2) and adipose-derived pericytes (ADPs) in a rat critical-sized calvarial defect model. In vitro analysis revealed that the HCF hydrogel was able to control the BMP-2 release and induce alkaline phosphatase (ALP) activity in neonatal rat osteoblasts. In addition, it was found that eluted BMP-2 significantly induced the osteogenic differentiation of ADPs. It was characterized by the increased ALP activity, osteocalcin expression and calcium deposits in ADPs. In vivo studies have shown that both HCF hydrogel with BMP-2 and HCF hydrogel with pericytes are able to significantly increase the regeneration of critical-sized calvarial defects in comparison with the control group. Nevertheless, the greatest regenerative effect was found after the co-delivery of ADPs and BMP-2 into a critical-sized calvarial defect. Thus, our findings suggest that the combined delivery of ADPs and BMP-2 in HCF hydrogel holds promise to be applied as an alternative biopolymer for the critical-sized bone defect restoration. [ABSTRACT FROM AUTHOR]

The treatment of critical‐sized bone defects has long been a major problem for surgeons. In this study, an intramedullary nail shaped three‐dimensional (3D)‐printed porous titanium implant that is capable of releasing strontium ions was developed through a simple and cost‐effective surface modification technique. The feasibility of this implant as a stand‐alone solution was evaluated using a rabbit's segmental diaphyseal as a defect model. The strontium‐loaded implant exhibited a favorable environment for cell adhesion, and mechanical properties that were commensurate with those of a rabbit's cortical bone. Radiographic, biomechanical, and histological analyses revealed a significantly higher amount of bone ingrowth and superior bone‐bonding strength in the strontium‐loaded implant when compared to an untreated porous titanium implant. Furthermore, one‐year histological observations revealed that the strontium‐loaded implant preserved the native‐like diaphyseal bone structure without failure. These findings suggest that strontium‐releasing 3D‐printed titanium implants have the clinical potential to induce the early and efficient repair of critical‐sized, load‐bearing bone defects. [ABSTRACT FROM AUTHOR]

The scarcity of native periosteum poses a significant clinical barrier in the repair of critical-sized bone defects. The challenge of enhancing regenerative potential in bone healing is further compounded by oxidative stress at the fracture site. However, the introduction of artificial periosteum has demonstrated its ability to promote bone regeneration through the provision of appropriate mechanical support and controlled release of pro-osteogenic factors. In this study, a poly (l -lactic acid) (PLLA)/hyaluronic acid (HA)-based nanofibrous membrane was fabricated using the coaxial electrospinning technique. The incorporation of irisin into the core-shell structure of PLLA/HA nanofibers (PLLA/HA@Irisin) achieved its sustained release. In vitro experiments demonstrated that the PLLA/HA@Irisin membranes exhibited favorable biocompatibility. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) was improved by PLLA/HA@Irisin, as evidenced by a significant increase in alkaline phosphatase activity and matrix mineralization. Mechanistically, PLLA/HA@Irisin significantly enhanced the mitochondrial function of BMMSCs via the activation of the sirtuin 3 antioxidant pathway. To assess the therapeutic effectiveness, PLLA/HA@Irisin membranes were implanted in situ into critical-sized calvarial defects in rats. The results at 4 and 8 weeks post-surgery indicated that the implantation of PLLA/HA@Irisin exhibited superior efficacy in promoting vascularized bone formation, as demonstrated by the enhancement of bone matrix synthesis and the development of new blood vessels. The results of our study indicate that the electrospun PLLA/HA@Irisin nanofibers possess characteristics of a biomimetic periosteum, showing potential for effectively treating critical-sized bone defects by improving the mitochondrial function and maintaining redox homeostasis of BMMSCs. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to assess the potential of poly (acrylic acid)/tricalcium phosphate nanoparticles (PAA/triCaPNPs) scaffold in terms of biocompatibility and osteoconductivity properties the in-vivo evaluation as well as to investigate the performance of PAA/triCaPNPs scaffold (with or without exosomes derived from UC-MSCs) for bone regeneration of rat critical-sized defect. Methods: PAA/triCaPNPs scaffold was made from acrylic acid (AA) monomer, N,N’-methylenebisacrylamide (MBAA), sodium bicarbonate (SBC), and ammonium persulfate (APS) through freeze-drying method. For in vivo evaluation, we randomly divided 24 rats into three groups. The rat calvarial bone defects were treated as follows: (1) Control group: defects without any treatment, (2) scaffold group: defects treated with scaffold only, (3) scaffold+exo group: defects treated with scaffold enriched with exosomes (1 μg/μL, 150 μg per rat). Eight- and 12-weeks post-surgery, half of the animals were sacrificed and bone regeneration was examined through micro-computerized tomography (µ-CT), histological staining, and immunohistochemistry (IHC). Results: Quantitative analysis based on µ-CT scan images at 8 and 12 weeks post-implantation clearly indicated that healing rate for defects that were filled with scaffold enriched with exosome was significantly higher than defects filled with scaffold without exosome. The H&E and Masson staining results revealed that more new bone-like form developed in the scaffold+exo group than that in control and scaffold groups. Further, IHC staining for osteocalcin and CD31 confirmed that more bone healing in the scaffold+exo group at 12 weeks could be associated with osteogenesis and angiogenesis concurrently. Conclusion: In the present study, we aimed to investigate the therapeutic potential of PAA/triCaPNPs scaffold as a carrier of human UC-MSC-derived exosome to achieve the exosome-controlled release on calvarial bone defect. The in vivo results indicated that the exosome-enriched scaffold could effectively minify the defect area and improve the bone healing in rat model, and as such it could be an option for exosome-based therapy.

Additive manufacturing has received attention for the fabrication of medical implants that have customized and complicated structures. Biodegradable Zn metals are revolutionary materials for orthopedic implants. In this study, pure Zn porous scaffolds with diamond structures were fabricated using customized laser powder bed fusion (L-PBF) technology. First, the mechanical properties, corrosion behavior, and biocompatibility of the pure Zn porous scaffolds were characterized in vitro. The scaffolds were then implanted into the rabbit femur critical-size bone defect model for 24 weeks. The results showed that the pure Zn porous scaffolds had compressive strength and rigidity comparable to those of cancellous bone, as well as relatively suitable degradation rates for bone regeneration. A benign host response was observed using hematoxylin and eosin (HE) staining of the heart, liver, spleen, lungs, and kidneys. Moreover, the pure Zn porous scaffold showed good biocompatibility and osteogenic promotion ability in vivo. This study showed that pure Zn porous scaffolds with customized structures fabricated using L-PBF represent a promising biodegradable solution for treating large bone defects.

Critical-sized bone defects resulting from trauma, inflammation, and tumor resections are individual in their size and shape. Implants for the treatment of such defects have to consider biomechanical and biomedical factors, as well as the individual conditions within the implantation site. In this context, 3D printing technologies offer new possibilities to design and produce patient-specific implants reflecting the outer shape and internal structure of the replaced bone tissue. The selection or modification of materials used in 3D printing enables the adaption of the implant, by enhancing the osteoinductive or biomechanical properties. In this study, scaffolds with bone spongiosa-inspired structure for extrusion-based 3D printing were generated. The computer aided design process resulted in an up scaled and simplified version of the bone spongiosa. To enhance the osteoinductive properties of the 3D printed construct, polycaprolactone (PCL) was combined with 20% (wt) calcium phosphate nano powder (CaP). The implants were designed in form of a ring structure and revealed an irregular and interconnected porous structure with a calculated porosity of 35.2% and a compression strength within the range of the natural cancellous bone. The implants were assessed in terms of biocompatibility and osteoinductivity using the osteosarcoma cell line MG63 and patient-derived mesenchymal stem cells in selected experiments. Cell growth and differentiation over 14 days were monitored using confocal laser scanning microscopy, scanning electron microscopy, deoxyribonucleic acid (DNA) quantification, gene expression analysis, and quantitative assessment of calcification. MG63 cells and human mesenchymal stem cells (hMSC) adhered to the printed implants and revealed a typical elongated morphology as indicated by microscopy. Using DNA quantification, no differences for PCL or PCL-CaP in the initial adhesion of MG63 cells were observed, while the PCL-based scaffolds favored cell proliferation in the early phases of culture up to 7 days. In contrast, on PCL-CaP, cell proliferation for MG63 cells was not evident, while data from PCR and the levels of calcification, or alkaline phosphatase activity, indicated osteogenic differentiation within the PCL-CaP constructs over time. For hMSC, the highest levels in the total calcium content were observed for the PCL-CaP constructs, thus underlining the osteoinductive properties.

The repair of critical-sized calvarial defects is a challenging problem for orthopedic surgery. One of the promising strategies of bone bioengineering to enhance the efficacy of large bone defect regeneration is the combined delivery of stem cells with osteoinductive factors within polymer carriers. The purpose of the research was to study the regenerative effects of heparin-conjugated fibrin (HCF) hydrogel containing bone morphogenetic protein 2 (BMP-2) and adipose-derived pericytes (ADPs) in a rat critical-sized calvarial defect model. In vitro analysis revealed that the HCF hydrogel was able to control the BMP-2 release and induce alkaline phosphatase (ALP) activity in neonatal rat osteoblasts. In addition, it was found that eluted BMP-2 significantly induced the osteogenic differentiation of ADPs. It was characterized by the increased ALP activity, osteocalcin expression and calcium deposits in ADPs. In vivo studies have shown that both HCF hydrogel with BMP-2 and HCF hydrogel with pericytes are able to significantly increase the regeneration of critical-sized calvarial defects in comparison with the control group. Nevertheless, the greatest regenerative effect was found after the co-delivery of ADPs and BMP-2 into a critical-sized calvarial defect. Thus, our findings suggest that the combined delivery of ADPs and BMP-2 in HCF hydrogel holds promise to be applied as an alternative biopolymer for the critical-sized bone defect restoration.

Abstract Background Repair of large-sized bone defects is a challengeable obstacle in orthopedics and evoked the demand for the development of biomaterials that could induce bone repair in such defects. Recently, UiO-66 has emerged as an attractive metal–organic framework (MOF) nanostructure that is incorporated in biomedical applications due to its biocompatibility, porosity, and stability. In addition, its osteogenic properties have earned a great interest as a promising field of research. Thus, the UiO-66 was prepared in this study and assessed for its potential to stimulate and support osteogenesis in vitro and in vivo in a rabbit femoral condyle defect model. The nanomaterial was fabricated and characterized using x-ray diffraction (XRD) and transmission electron microscopy (TEM). Afterward, in vitro cytotoxicity and hemolysis assays were performed to investigate UiO-66 biocompatibility. Furthermore, the material in vitro capability to upregulate osteoblast marker genes was assessed using qPCR. Next, the in vivo new bone formation potential of the UiO-66 nanomaterial was evaluated after induction of bone defects in rabbit femoral condyles. These defects were left empty or filled with UiO-66 nanomaterial and monitored at weeks 4, 8, and 12 after bone defect induction using x-ray, computed tomography (CT), histological examinations, and qPCR analysis of osteocalcin (OC) and osteopontin (OP) expressions. Results The designed UiO-66 nanomaterial showed excellent cytocompatibility and hemocompatibility and stimulated the in vitro osteoblast functions. The in vivo osteogenesis was enhanced in the UiO-66 treated group compared to the control group, whereas evidence of healing of the treated bone defects was observed grossly and histologically. Interestingly, UiO-66 implanted defects displayed a significant osteoid tissue and collagen deposition compared to control defects. Moreover, the UiO-66 nanomaterial demonstrated the potential to upregulate OC and OP in vivo. Conclusions The UiO-66 nanomaterial implantation possesses a stimulatory impact on the healing process of critical-sized bone defects indicating that UiO-66 is a promising biomaterial for application in bone tissue engineering.

The critical-sized bone defect resulting from trauma, tumor resection, and congenital deformity fails to undergo spontaneous healing due to its substantial size, while the ensuing inflammatory process and hypoxic environment further impede the regenerative process. Therefore, it has consistently presented a significant clinical challenge. In the present study, we incorporate a glycyrrhizic acid (GA)-functionalized hydrogel onto the surface of a Hydroxyapatite (Hap)-modified Polycaprolactone (PCL) scaffold to fabricate a composite scaffold. The composed scaffold showed favorable anti-inflammatory and antioxidative capabilities by modulating macrophage polarization and scavenging reactive oxygen species (ROS); the modification of Hap enhanced its osteogenic ability. An in vivo rat skull defect model confirmed that the composed scaffold efficiently promotes bone regeneration. In general, the composed scaffold with the ability of osteoimmune microenvironment regulation can effectively repair critical-sized bone defects. This strategy provides a promising method for the reconstruction of large segmental bone defects.

A novel biodegradable metal system, ZnLiCa ternary alloys, were systematically investigated both in vitro and in vivo. The ultimate tensile strength (UTS) of Zn0.8Li0.1Ca alloy reached 567.60 ± 9.56 MPa, which is comparable to pure Ti, one of the most common material used in orthopedics. The elongation of Zn0.8Li0.1Ca is 27.82 ± 18.35%, which is the highest among the ZnLiCa alloys. The in vitro degradation rate of Zn0.8Li0.1Ca alloy in simulated body fluid (SBF) showed significant acceleration than that of pure Zn. CCK-8 tests and hemocompatibility tests manifested that ZnLiCa alloys exhibit good biocompatibility. Real-time PCR showed that Zn0.8Li0.1Ca alloy successfully stimulated the expressions of osteogenesis-related genes (ALP, COL-1, OCN and Runx-2), especially the OCN. An in vivo implantation was conducted in the radius of New Zealand rabbits for 24 weeks, aiming to treat the bone defects. The Micro-CT and histological evaluations proved that the regeneration of bone defect was faster within the Zn0.8Li0.1Ca alloy scaffold than the pure Ti scaffold. Zn0.8Li0.1Ca alloy showed great potential to be applied in orthopedics, especially in the load-bearing sites.

In craniofacial bone defects, the promotion of bone volume augmentation remains a challenge. Finding strategies for bone regeneration such as combining resorbable minerals with organic polymers would contribute to solving the bone volume roadblock. Here, dicalcium phosphate dihydrate, chitosan and hyaluronic acid were used to functionalize a bone-side collagen membrane. Despite an increase in the release of inflammatory mediators by human circulating monocytes, the in vivo implantation of the functionalized membrane allowed the repair of a critical-sized defect in a calvaria rat model with de novo bone exhibiting physiological matrix composition and structural organization. Microtomography, histological and Raman analysis combined with nanoindentation testing revealed an increase in bone volume in the presence of the functionalized membrane and the formation of woven bone after eight weeks of implantation; these data showed the potential of dicalcium phosphate dihydrate, chitosan and hyaluronic acid to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in animal models. [ABSTRACT FROM AUTHOR]

BACKGROUND: The bone scaffold with complex porous structure can be customized accurately and individually by additive manufacturing technology, so as to achieve the dual bionics of the structure and function of cancellous bone at critical-sized bone defects. OBJECTIVE: To clear the mechanical properties and biological activity of β-tricalcium phosphate bioceramic bone scaffold with triply periodic minimal surface structure, and reveal the regulatory effect of triply periodic minimal surface structure on osteoblasts through material and cytological characterizations. METHODS: Three kinds of triply periodic minimal surface G surface structure β-tricalcium phosphate bioceramic bone scaffolds with different pore sizes of 330, 420, and 510 μm were designed by Matlab R2020a software, and the design drawings were analyzed by Inspire 2018 software. The STL file exported based on the triply periodic minimal surface structure was the blueprint. β-Tricalcium phosphate scaffolds were fabricated by additive manufacturing technology based on digital laser processing. The surface morphology was observed by scanning electron microscope. The phase composition was detected by X-ray diffractometer, and the mechanical strength was detected by universal material testing machine. MC3T3-E1 cells were co-cultured with scaffolds. Cell proliferation, cell adhesion, and alkaline phosphatase activity were detected. RESULTS AND CONCLUSION: (1) Inspire 2018 software showed that triply periodic minimal surface presented a smooth, continuous and uniform porous structure. (2) Scanning electron microscope confirmed that the additive manufacturing technology based on digital laser processing successfully realized the accurate molding of triply periodic minimal surface structure. (3) X-ray diffraction confirmed that the scaffold was composed of pure β-tricalcium phosphate crystal phase. (4) The compressive strength and elastic modulus of the three groups of scaffolds were all within or near the range of cancellous bone, and the compressive strength of the scaffolds was inversely proportional to the pore size. (5) CCK8 assay showed that MC3T3-E1 cells grew well on the scaffolds; the biological activity of the scaffolds was dose-dependent with the pore size; and the 510 μm pore diameter scaffold had the best effect on promoting proliferation. (6) Living cell imager and laser confocal microscope showed that MC3T3-E1 cells could adhere to the scaffold in the early stage, and the amount of adhesion increased with the increase of pore size. (7) Alkaline phosphatase activity analysis showed that the alkaline phosphatase activity of cells on 420 μm scaffold was the highest. (8) The results show that the mechanical properties and biological activity of the triply periodic minimal surface structure β-tricalcium phosphate bioceramic scaffold are excellent; the pore size of 420 μm is beneficial to cell differentiation and 510 μm is beneficial to cell proliferation, which has the potential to repair critical bone defects. [ABSTRACT FROM AUTHOR]

Background: Repair of large-sized bone defects is a challengeable obstacle in orthopedics and evoked the demand for the development of biomaterials that could induce bone repair in such defects. Recently, UiO-66 has emerged as an attractive metal–organic framework (MOF) nanostructure that is incorporated in biomedical applications due to its biocompatibility, porosity, and stability. In addition, its osteogenic properties have earned a great interest as a promising field of research. Thus, the UiO-66 was prepared in this study and assessed for its potential to stimulate and support osteogenesis in vitro and in vivo in a rabbit femoral condyle defect model. The nanomaterial was fabricated and characterized using x-ray diffraction (XRD) and transmission electron microscopy (TEM). Afterward, in vitro cytotoxicity and hemolysis assays were performed to investigate UiO-66 biocompatibility. Furthermore, the material in vitro capability to upregulate osteoblast marker genes was assessed using qPCR. Next, the in vivo new bone formation potential of the UiO-66 nanomaterial was evaluated after induction of bone defects in rabbit femoral condyles. These defects were left empty or filled with UiO-66 nanomaterial and monitored at weeks 4, 8, and 12 after bone defect induction using x-ray, computed tomography (CT), histological examinations, and qPCR analysis of osteocalcin (OC) and osteopontin (OP) expressions. Results: The designed UiO-66 nanomaterial showed excellent cytocompatibility and hemocompatibility and stimulated the in vitro osteoblast functions. The in vivo osteogenesis was enhanced in the UiO-66 treated group compared to the control group, whereas evidence of healing of the treated bone defects was observed grossly and histologically. Interestingly, UiO-66 implanted defects displayed a significant osteoid tissue and collagen deposition compared to control defects. Moreover, the UiO-66 nanomaterial demonstrated the potential to upregulate OC and OP in vivo. Conclusions: The UiO-66 nanomaterial implantation possesses a stimulatory impact on the healing process of critical-sized bone defects indicating that UiO-66 is a promising biomaterial for application in bone tissue engineering. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSC) can be used to facilitate reparative osteogenesis. In the case of critical-size defects, MSC can attach to allogenic bone implants (AlloI) that serve as a matrix. Objective. Analyze the morphological features of reparative osteogenesis in critical-size defects in femurs of rats (3 and 12 months old) when the defects are filled with MSC along with AlloI. Methods. 60 white lab rats, 3 months (n=30) and 12 months (n=30) old were used. Defects (3mm in depth, 3mm in diameter) were created in the femoral metaphysis of each rat, and filled with AlloI in the control groups and with AlloI and adipose-derived MSC in the experimental groups. Each group contained 15 rats of a particular age. 14, 28, and 90 days after the surgery, histological studies were conducted. Results. The area of AlloI decreased with time. 14 days after the surgery, in the experimental group, the area of AlloI was 1.6 times greater in 3-month-old (3mo) rats than in 12-month-old (12mo) rats. In comparison to the control, the area of AlloI was greater 14 days after surgery in 3mo rats and 28 days after surgery in 12mo rats. 14 and 28 days after the operation, the area of connective tissue was greater in rats of both experimental groups than in the control. For the 3mo rats, the same was true 90 days after the operation. The area of newly formed bone was 1.6 times lower in 3mo rats than in 12mo rats 14 days after the operation. 90 days after the operation, the area was 2.3 greater in 3mo rats. For 12mo rats, the highest area of bone tissue occurred 14 days after the surgery, and subsequently did not significantly change or differ from the control. For 3mo rats, the area of bone tissue was lower than control 14 and 28 days after the surgery, but greater than control 90 days after the surgery. Conclusions. The use of MSC along with AlloI to fill traumatic bone defects causes slower bone formation and excessive formation of connective tissue, independent of the age of the recipient. [ABSTRACT FROM AUTHOR]

In vivo bioreactors serve as regenerative niches that improve vascularization and regeneration of bone grafts. This study has evaluated the masseter muscle as a natural bioreactor for βTCP or PCL/βTCP scaffolds, in terms of bone regeneration. The effect of pedicle preservation, along with sole, or MSC‐ or rhBMP2‐combined application of scaffolds, has also been studied. Twenty‐four mongrel dogs were randomly placed in six groups, including βTCP, βTCP/rhBMP2, βTCP/MSCs, PCL/βTCP, PCL/βTCP/rhBMP2, and PCL/βTCP/MSCs. During the first surgery, the scaffolds were implanted into the masseter muscle for being prefabricated. After 2 months, each group was divided into two subgroups prior to mandibular bone defect reconstruction; one with a preserved vascularized pedicle and one without. After 12 weeks, animals were euthanized, and new bone formation was evaluated using histological analysis. Histological analysis showed that all β‐TCP scaffold groups had resulted in significantly greater rates of new bone formation, either with a pedicle surgical approach or non‐pedicle surgical approach, comparing to their parallel groups of βTCP/PCL scaffolds (p ≤.05). Pedicled β‐TCP scaffold groups that were treated with either rhBMP2 (48.443% ± 0.250%) or MSCs (46.577% ± 0.601%) demonstrated the highest rates of new bone formation (p ≤.05). Therefore, masseter muscle can be used as a local in vivo bioreactor with potential clinical advantages in reconstruction of human mandibular defects. In addition, scaffold composition, pedicle preservation, and treatment with MSCs or rhBMP2, influence new bone formation and scaffold degradation rates in the prefabrication technique. [ABSTRACT FROM AUTHOR]

Abstract Background Due to their multilineage potential and high proliferation rate, mesenchymal stem cells (MSC) indicate a sufficient alternative in regenerative medicine. In comparison to the commonly used 2-dimensional culturing method, culturing cells as spheroids stimulates the cell-cell communication and mimics the in vivo milieu more accurately, resulting in an enhanced regenerative potential. To investigate the osteoregenerative potential of MSC spheroids in comparison to MSC suspensions, cell-loaded fibrin gels were implanted into murine critical-sized femoral bone defects. Methods After harvesting MSCs from 4 healthy human donors and preculturing and immobilizing them in fibrin gel, cells were implanted into 2 mm murine femoral defects and stabilized with an external fixator. Therefore, 26 14- to 15-week-old nu/nu NOD/SCID nude mice were randomized into 2 groups (MSC spheroids, MSC suspensions) and observed for 6 weeks. Subsequently, micro-computed tomography scans were performed to analyze regenerated bone volume and bone mineral density. Additionally, histological analysis, evaluating the number of osteoblasts, osteoclasts and vessels at the defect side, were performed. Statistical analyzation was performed by using the Student’s t-test and, the Mann-Whitney test. The level of significance was set at p = 0.05. Results μCT-analysis revealed a significantly higher bone mineral density of the MSC spheroid group compared to the MSC suspension group. However, regenerated bone volume of the defect side was comparable between both groups. Furthermore, no significant differences in histological analysis between both groups could be shown. Conclusion Our in vivo results reveal that the osteo-regenerative potential of MSC spheroids is similar to MSC suspensions.

Abstract Background The healing of large critical-sized bone defects remains a clinical challenge in modern orthopedic medicine. The current gold standard for treating critical-sized bone defects is autologous bone graft; however, it has critical limitations. Bone tissue engineering has been proposed as a viable alternative, not only for replacing the current standard treatment, but also for producing complete regeneration of bone tissue without complex surgical treatments or tissue transplantation. In this study, we proposed a transplantable radially patterned scaffold for bone regeneration that was defined by capillary force lithography technology using biodegradable polycaprolactone polymer. Results The radially patterned transplantable biodegradable scaffolds had a radial structure aligned in a central direction. The radially aligned pattern significantly promoted the recruitment of host cells and migration of osteoblasts into the defect site. Furthermore, the transplantable scaffolds promoted regeneration of critical-sized bone defects by inducing cell migration and differentiation. Conclusions Our findings demonstrated that topographically defined radially patterned transplantable biodegradable scaffolds may have great potential for clinical application of bone tissue regeneration.

Abstract Background Critical-sized bone defects, mainly from trauma, infection or tumor resection are a challenging condition, often resulting in prolonged, complicated course of treatment. Autografts are considered as the gold standard to replace lost bone. However, limited amount of bone graft volume and donor-site morbidity have established the need for the development of alternative methods such as scaffold-based tissue engineering (TE). The emerging market of additive manufacturing (3D-printing) has markedly influenced the manufacturing of scaffolds out of a variety of biodegradable materials. Particularly medical-grade polycaprolactone and tricalcium phosphate (mPCL–TCP) scaffolds show appropriate biocompatibility and osteoconduction with good biomechanical strength in large preclinical animal models. This case report aims to show first evidence of the feasibility, safety, and efficacy of mPCL–TCP scaffolds applied in a patient with a long bone segmental defect. Case presentation The presented case comprises a 29-year-old patient who has suffered a left-sided II° open femoral shaft fracture. After initial external fixation and subsequent conversion to reamed antegrade femoral nailing, the patient presented with an infection in the area of the formerly open fracture. Multiple revision surgeries followed to eradicate microbial colonization and attempt to achieve bone healing. However, 18 months after the index event, still insufficient diaphyseal bone formation was observed with circumferential bony defect measuring 6 cm at the medial and 11 cm at the lateral aspect of the femur. Therefore, the patient received a patient-specific mPCL–TCP scaffold, fitting the exact anatomical defect and the inserted nail, combined with autologous bone graft (ABG) harvested with the Reamer–Irrigator–Aspirator system (RIA—Synthes®) as well as bone morphogenetic protein-2 (BMP-2). Radiographic follow-up 12 months after implantation of the TE scaffold shows advanced bony fusion and bone formation inside and outside the fully interconnected scaffold architecture. Conclusion This case report shows a promising translation of scaffold-based TE from bench to bedside. Preliminary evidence indicates that the use of medical-grade scaffolds is safe and has the potential to improve bone healing. Further, its synergistic effects when combined with ABG and BMP-2 show the potential of mPCL–TCP scaffolds to support new bone formation in segmental long bone defects.

Although autogenous bone grafts are an optimal filling material for the induced membrane technique, limited availability and complications at the harvest site have created a need for alternative graft materials. We aimed to investigate the effect of an rhBMP‐2‐coated, 3D‐printed, macro/microporous CaO–SiO2–P2O5–B2O3 bioactive ceramic scaffold in the treatment of critical femoral bone defects in rabbits using the induced membrane technique. A 15‐mm segmental bone defect was made in the metadiaphyseal area of the distal femur of 14 rabbits. The defect was filled with polymethylmethacrylate cement and stabilized with a 2.0 mm locking plate. After the membrane matured for 4 weeks, the scaffold was implanted in two randomized groups: Group A (3D‐printed bioceramic scaffold) and Group B (3D‐printed, bioceramic scaffold with rhBMP‐2). Eight weeks after implantation, the radiographic assessment showed that the healing rate of the defect was significantly higher in Group B (7/7, 100%) than in Group A (2/7, 29%). The mean volume of new bone formation around and inside the scaffold doubled in Group B compared to that in Group A. The mean static and dynamic stiffness were significantly higher in Group B. Histological examination revealed newly formed bone in both groups. Extensive cortical bone formation along the scaffold was found in Group B. Successful bone reconstruction in critical‐sized bone defects could be obtained using rhBMP‐2‐coated, 3D‐printed, macro/microporous bioactive ceramic scaffolds. This grafting material demonstrated potential as an alternative graft material in the induced membrane technique for reconstructing critical‐sized bone defects. [ABSTRACT FROM AUTHOR]

Several synthetic and natural scaffolds have been developed for bone tissue engineering (BTE). This study was designed to fabricate and characterize a composite scaffold based on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV)/polycaprolactone (PCL) polymers containing 5 % Hydroxyapatite (HA) nano-particles (HA5) for regeneration of bone critical size defect in a rat animal model. In the first step of this study, the PHBV/PCL (30 and 70 wt.%, respectively) with and without HA nanoparticle scaffolds were fabricated by Dual-Leaching Technique and then evaluated for FTIR spectroscopy, SEM, degradation rate, cell binding and proliferation. In the second step, the scaffolds were applied for cell differentiation and animal study. Human osteoblast-like Saos-2 cell line was seeded onto the scaffolds and Alkaline Phosphatase (ALP) activity was assayed on 7 days and 14 post-culture. The capacity of scaffolds to promote the bone regeneration genes was also investigated using quantitative RT-PCR. The critical size defects were made on the dorsal part of the rat's calvarium and were filled with PCL70PHBV30 and PCL70PHBV30HA5 scaffolds and were then evaluated histologically and histomorphometrically. The higher mean percentage of new bone formation, bone cells and osteon, as well as lower fibrous connective tissue, was detected in the PCL70PHBV30HA5 group than that of the other groups. In conclusion, the HA nanoparticles at 5 % concentration have been well distributed in the PCL70PHBV30 scaffold that could accelerate bone regeneration. [ABSTRACT FROM AUTHOR]

In craniofacial bone defects, the promotion of bone volume augmentation remains a challenge. Finding strategies for bone regeneration such as combining resorbable minerals with organic polymers would contribute to solving the bone volume roadblock. Here, dicalcium phosphate dihydrate, chitosan and hyaluronic acid were used to functionalize a bone-side collagen membrane. Despite an increase in the release of inflammatory mediators by human circulating monocytes, the in vivo implantation of the functionalized membrane allowed the repair of a critical-sized defect in a calvaria rat model with de novo bone exhibiting physiological matrix composition and structural organization. Microtomography, histological and Raman analysis combined with nanoindentation testing revealed an increase in bone volume in the presence of the functionalized membrane and the formation of woven bone after eight weeks of implantation; these data showed the potential of dicalcium phosphate dihydrate, chitosan and hyaluronic acid to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in animal models.

Introduction: The management of acquired long bone defects (LBDs) is a persistent problem in modern orthopedics with few effective solutions. The goal of this review is to conduct a thorough analysis of existing diaphyseal LBD classifications and to identify if any of these may be adopted for universal application in future. Materials and Methods: A literature review was performed analyzing articles between 1970 and 2019 on PubMed (English) and CyberLeninka (Russian) databases. All studies developing or utilizing a classification tool were eligible for inclusion. The search retrieved a total of 1032 articles from PubMed, 477 from CyberLennika, and 28 articles from reference lists. Any disagreement regarding the inclusion of articles was resolved by consensus between all authors. Results: A total of 18 classifications in the literature underwent analysis. Among the classifications we analyzed, 12 described posttraumatic defects, 6 described osteomyelitis defects or infected nonunions, and 5 described, among other defects, iatrogenic bone. Of the 18 classifications, 12 have a focus on a specific treatment method. We found that consensus exists between most authors regarding the criteria necessary to measure to determine the most appropriate treatment method. These key factors include the size and location of the bone defect. Conclusion: This analysis showed that despite the large number of publications devoted to the classification and treatment of LBD, there is still no consensus among authors regarding not only a universal clinical classification but also a general definition of the term "bone defect." The classification proposed by Solomin et al. is very useful but in its present form, has significant drawbacks and requires further improvement before it can be adopted as a universal classification system for LBDs. [ABSTRACT FROM AUTHOR]

Background: Due to their multilineage potential and high proliferation rate, mesenchymal stem cells (MSC) indicate a sufficient alternative in regenerative medicine. In comparison to the commonly used 2-dimensional culturing method, culturing cells as spheroids stimulates the cell-cell communication and mimics the in vivo milieu more accurately, resulting in an enhanced regenerative potential. To investigate the osteoregenerative potential of MSC spheroids in comparison to MSC suspensions, cell-loaded fibrin gels were implanted into murine critical-sized femoral bone defects.Methods: After harvesting MSCs from 4 healthy human donors and preculturing and immobilizing them in fibrin gel, cells were implanted into 2 mm murine femoral defects and stabilized with an external fixator. Therefore, 26 14- to 15-week-old nu/nu NOD/SCID nude mice were randomized into 2 groups (MSC spheroids, MSC suspensions) and observed for 6 weeks. Subsequently, micro-computed tomography scans were performed to analyze regenerated bone volume and bone mineral density. Additionally, histological analysis, evaluating the number of osteoblasts, osteoclasts and vessels at the defect side, were performed. Statistical analyzation was performed by using the Student's t-test and, the Mann-Whitney test. The level of significance was set at p = 0.05.Results: μCT-analysis revealed a significantly higher bone mineral density of the MSC spheroid group compared to the MSC suspension group. However, regenerated bone volume of the defect side was comparable between both groups. Furthermore, no significant differences in histological analysis between both groups could be shown.Conclusion: Our in vivo results reveal that the osteo-regenerative potential of MSC spheroids is similar to MSC suspensions. [ABSTRACT FROM AUTHOR]

Background: The healing of large critical-sized bone defects remains a clinical challenge in modern orthopedic medicine. The current gold standard for treating critical-sized bone defects is autologous bone graft; however, it has critical limitations. Bone tissue engineering has been proposed as a viable alternative, not only for replacing the current standard treatment, but also for producing complete regeneration of bone tissue without complex surgical treatments or tissue transplantation. In this study, we proposed a transplantable radially patterned scaffold for bone regeneration that was defined by capillary force lithography technology using biodegradable polycaprolactone polymer. Results: The radially patterned transplantable biodegradable scaffolds had a radial structure aligned in a central direction. The radially aligned pattern significantly promoted the recruitment of host cells and migration of osteoblasts into the defect site. Furthermore, the transplantable scaffolds promoted regeneration of critical-sized bone defects by inducing cell migration and differentiation. Conclusions: Our findings demonstrated that topographically defined radially patterned transplantable biodegradable scaffolds may have great potential for clinical application of bone tissue regeneration. [ABSTRACT FROM AUTHOR]

Introduction: The current 'gold-standard' treatment of critical-sized bone defects (CSBDs) is autografts; however, they have drawbacks including lack of massive bone source donor site morbidity, incomplete remodeling, and the risk of infection. One potential treatment for treating CSBDs is bone marrow-derived mesenchymal stem cells (BM-MSCs). Previously, there were no studies regarding the use of human umbilical cord-mesenchymal stem cells (hUC-MSCs) for treating BDs. We aim to investigate the use of allogeneic hUC-MSCs for treating CSBDs. Method: We included subjects who were diagnosed with non-union fracture with CSBDs who agreed to undergo hUC-MSCs implantation. All patients were given allogeneic hUC-MSCs. All MSCs were obtained and cultured using the multiple-harvest explant method. Subjects were evaluated functionally using the Lower Extremity Functional Scale (LEFS) and radiologically by volume defect reduction. Result: A total of seven (3 male, 4 female) subjects were recruited for this study. The subjects age ranged from 14 to 62 years. All seven subjects had increased LEFS during the end of the follow-up period, indicating improved functional ability. The follow-up period ranged from 12 to 36 months. One subject had wound dehiscence and infection, and two subjects developed partial union. Conclusion: Umbilical cord mesenchymal stem cells are a potential new treatment for CSBDs. Additional studies with larger samples and control groups are required to further investigate the safety and efficacy of umbilical cord-derived mesenchymal stem cells for treating CSBDs. [ABSTRACT FROM AUTHOR]

Adequate bone tissue regeneration has been challenging to achieve at critical-sized bone defects caused by disease. Bone tissue engineering using a combination of scaffolds and bioactive factors provides new hope for the treatment of this extreme condition. Icaritin, a herb-derived chemical, has shown its ability to enhance bone formation both in vitro and in vivo, and it has been found that sub-micron surface structure instructs bone formation in calcium phosphate ceramics (CaPs). Here, we evaluated the possibility of using a submicron surface structured CaP ceramic as the carrier of icaritin for bone tissue regeneration in critical-sized bone defects. Icaritin, an herb-derived chemical, was loaded into a submicron surface structured porous calcium phosphate ceramic (Ø12.8 × 3 mm) to get samples with 0, 10, 50, 250, and 1,250 µg icaritin per CaP disc (M0, M10, M50, M250, M1250 groups, respectively). In vitro evaluation with the certain dosages correlated to those released from the samples showed a dose-dependent enhancement of osteogenic differentiation and mineralization of human bone marrow stromal cells with the presence of osteogenic factors in the culture medium, indicating icaritin is an osteopromotive factor. After intramuscular implantation of the samples in dogs for 8 weeks, a dose-dependent of bone formation was seen with enhanced bone formation at the dosage of 50 and 250 µg. To evaluate the in vivo osteogenic potentials of icaritin-containing CaP ceramic scaffolds in the orthopedic site, a 12.8 mm calvarial defect model in rabbits was established. Micro-computed tomography (micro-CT) and histology results at weeks 4, 8 and 12 post-surgery showed more newly formed bone in M250 group, with correspondingly more new vessel ingrowth. The results presented herein suggested that being osteopromotive, icaritin could enhance bone formation initiated by sub-microstructured CaP ceramics and the CaP ceramics scaffold incorporating icaritin is a promising biomaterial for the treatment of critical-sized defect.

Background: Critical-sized bone defects, mainly from trauma, infection or tumor resection are a challenging condition, often resulting in prolonged, complicated course of treatment. Autografts are considered as the gold standard to replace lost bone. However, limited amount of bone graft volume and donor-site morbidity have established the need for the development of alternative methods such as scaffold-based tissue engineering (TE). The emerging market of additive manufacturing (3D-printing) has markedly influenced the manufacturing of scaffolds out of a variety of biodegradable materials. Particularly medical-grade polycaprolactone and tricalcium phosphate (mPCL–TCP) scaffolds show appropriate biocompatibility and osteoconduction with good biomechanical strength in large preclinical animal models. This case report aims to show first evidence of the feasibility, safety, and efficacy of mPCL–TCP scaffolds applied in a patient with a long bone segmental defect. Case presentation: The presented case comprises a 29-year-old patient who has suffered a left-sided II° open femoral shaft fracture. After initial external fixation and subsequent conversion to reamed antegrade femoral nailing, the patient presented with an infection in the area of the formerly open fracture. Multiple revision surgeries followed to eradicate microbial colonization and attempt to achieve bone healing. However, 18 months after the index event, still insufficient diaphyseal bone formation was observed with circumferential bony defect measuring 6 cm at the medial and 11 cm at the lateral aspect of the femur. Therefore, the patient received a patient-specific mPCL–TCP scaffold, fitting the exact anatomical defect and the inserted nail, combined with autologous bone graft (ABG) harvested with the Reamer–Irrigator–Aspirator system (RIA—Synthes®) as well as bone morphogenetic protein-2 (BMP-2). Radiographic follow-up 12 months after implantation of the TE scaffold shows advanced bony fusion and bone formation inside and outside the fully interconnected scaffold architecture. Conclusion: This case report shows a promising translation of scaffold-based TE from bench to bedside. Preliminary evidence indicates that the use of medical-grade scaffolds is safe and has the potential to improve bone healing. Further, its synergistic effects when combined with ABG and BMP-2 show the potential of mPCL–TCP scaffolds to support new bone formation in segmental long bone defects. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate whether porous beads with a leaf-stacked scaffold loaded with bone morphogenetic protein-2, with sustained release for up to four weeks, were beneficial to a dog model of critical long bone defects and capable of promoting bone regeneration without side effects. Critical long bone defects were created in the femoral diaphyses of 21 dogs. Each critical defect was fixed with an intramedullary pin and an universal locking plate. Leaf-stacked scaffold beads were implanted into the defect, which was covered with a membrane. Nineteen samples were evaluated 4, 8, and 12 weeks after surgery using simple radiographs, serum chemistry measurements, histological staining, and gene expression analysis. Sustained bone morphogenetic protein-2 release from the leaf-stacked scaffold continued for four weeks and consistently affected the expression of early and late osteogenesis factors. The results suggest that sustained bone morphogenetic protein-2 release is effective for bone regeneration, with minimal side effects. [ABSTRACT FROM AUTHOR]

Repair of critical-size bone defects (CBD) remains a challenge in orthopedic surgery due to the restricted regenerative capacity of bone tissue. Herein, multi-functional biphasic calcium phosphate (BCP) blocks decorated with gelatin packs were developed for bone regeneration of CBDs. The gelatin packs consist of gelatin microspheres (GMSs) and an enzymatic crosslinked gelatin layer for bone morphogenetic protein-2 (BMP-2) and antibiotics delivery. The porous interior of the BCP block was filled with GMSs containing BMP-2 and tightly packed with an enzymatic crosslinked gelatin layer. The outermost part of the block was then covered with antibiotics-loaded GMSs. The BCP platform elaborately decorated for each zone improved the adhesion of osteoblasts and enabled the sequential release of antibiotics and BMP-2 and sustained release of BMP-2, realizing on-demand drug release according to the treatment stage. The programmed drug release of this platform significantly improved the regeneration of CBDs. In vivo experiments using dog's mandible defect models clearly demonstrated the clinical application potential of this BCP platform. [Display omitted] • A bone graft functionalized with gelatin packs was developed. • The gelatin pack consists of gelatin microspheres (GMSs) optimized for the delivery of antibiotics and BMP-2. • This platform achieved programmed dual drug release to enable a one-step treatment of critical-sized bone defects. • This platform minimized the initial postoperative swelling and promoted accelerated bone formation. [ABSTRACT FROM AUTHOR]

Difficulties in treating pseudarthrosis and critical bone defects are still evident in physicians’ clinical routines. Bone morphogenetic protein 2 (BMP-2) has shown promising osteoinductive results but also considerable side effects, not unexpected given that it is a morphogen. Thus, the bone regenerative potential of the novel selective, non-morphogenic EP4 prostaglandin receptor agonist KMN-159 was investigated in this study. Therefore, mineralized collagen type-1 matrices were loaded with different amounts of BMP-2 or KMN-159 and implanted into a 5 mm critical-sized femoral defect in rats. After 12 weeks of observation, micro-computed tomography scans were performed to analyze the newly formed bone volume (BV) and bone mineral density (BMD). Histological analysis was performed to evaluate the degree of defect healing and the number of vessels, osteoclasts, and osteoblasts. Data were evaluated using Kruskal-Wallis followed by Dunn’s post hoc test. As expected, animals treated with BMP-2, the positive control for this model, showed a high amount of newly formed BV as well as bone healing. For KMN-159, a dose-dependent effect on bone regeneration could be observed up to a dose optimum, demonstrating that this non-morphogenic mechanism of action can stimulate bone formation in this model system.

Background/Aims: Human dental pulp-derived mesenchymal stromal cells (hDPSCs) are promising seed cells for tissue engineering due to their easy accessibility and multi-lineage differentiation. Pannexin3 (Panx3) plays crucial roles during bone development and differentiation. The aim of the present study was to investigate the effect of Panx3 on osteogenesis of hDPSCs and the underlying mechanism. Methods: Utilizing qRT-PCR, Western blot, and immunohistochemistry, we explored the change of Panx3 during osteogenic differentiation of hDPSCs. Next, hDPSCs with loss (Panx3 knockdown) and gain (Panx3 overexpression) of Panx3 function were developed to investigate the effects of Panx3 on osteogenic differentiation of hDPSC and the underlying mechanism. Finally, a commercial β-TCP scaffold carrying Panx3-modified hDPSCs was utilized to evaluate bone defect repair. Results: Panx3 was upregulated during osteogenic differentiation in a time-dependent manner. Panx3 overexpression promoted osteogenic differentiation of hDPSCs, whereas depletion of Panx3 resulted in a decline of differentiation, evidenced by upregulated expression of mineralization-related markers, increased alkaline phosphatase (ALP) activity, and enhanced ALP and Alizarin red staining. Panx3 was found to interact with the Wnt/β-catenin signaling pathway, forming a negative feedback loop. However, Wnt/β-catenin did not contribute to enhancement of osteogenic differentiation as observed in Panx3 overexpression. Moreover, Panx3 promoted osteogenic differentiation of hDPSCs via increasing ERK signaling pathway. Micro-CT and histological staining results showed that Panx3-modified hDPSCs significantly improved ossification of critical-sized bone defects. Conclusion: These findings suggest that Panx3 is a crucial modulator of hDPSCs differentiation.

Abstract Background Mesenchymal stem cells (MSCs) can be differentiated into an osteoblastic lineage in the presence of growth factors (GFs). Platelet-rich plasma (PRP), which can be easily isolated from whole blood, contains a large amount of GFs, and, therefore, promotes bone growth and regeneration. The main goal of this work was to develop and investigate the effect of a new sandwich-like bone scaffold which combines a nano-calcium sulfate (nCS) disc along with PRP fibrin gel (nCS/PRP) with BMP2-modified MSCs on bone repair and regeneration in rat critical-sized calvarial defects. Methods We evaluated the cytotoxicity, osteogenic differentiation and mineralization effect of PRP extract on BMP2-modified MSCs and constructed a sandwich-like nCS/PRP scaffold (mimicking the nano-calcium matrix of bone and carrying multi GFs in the PRP) containing BMP2-modified MSCs. The capacity of this multifunctional bone regeneration system in promoting bone repair was assessed in vivo in a rat critical-sized (8 mm) calvarial bone defect model. Results We developed an optimized nCS/PRP sandwich-like scaffold. Scanning electron microscopy (SEM) results showed that nCS/PRP are polyporous with an average pore diameter of 70–80 μm and the cells can survive in the nCS/PRP scaffold. PRP extract dramatically stimulated proliferation and differentiation of BMP2-modified MSCs in vitro. Our in vivo results showed that the combination of BMP2-modified MSCs and nCS/PRP scaffold dramatically increased new bone regeneration compared with the groups without PRP and/or BMP2. Conclusions nCS/PRP scaffolds containing BMP2-modified MSCs successfully promotes bone regeneration in critical-sized bone defects. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for critical-sized bone defects.

Icariin, a typical flavonol glycoside, is the main active component of Herba Epimedii, which was used to cure bone-related diseases in China for centuries. It has been reported that Icariin can be delivered locally by biomaterials and it has an osteogenic potential for bone tissue engineering. Biomimetic calcium phosphate (BioCaP) bone substitute is a novel drug delivery carrier system. Our study aimed to evaluate the osteogenic potential when Icariin was internally incorporated into the BioCaP granules. The BioCaP combined with Icariin and bone morphogenetic protein 2 (BMP-2) was investigated in vitro using an MC3T3-E1 cell line. We also investigated its efficacy to repair 8 mm diameter critical size bone defects in the skull of SD male rats. BioCaP was fabricated according to a well-established biomimetic mineralization process. In vitro, the effects of BioCaP alone or BioCaP with Icariin and/or BMP-2 on cell proliferation and osteogenic differentiation of MC3T3-E1 cells were systematically evaluated. In vivo, BioCaP alone or BioCaP with Icariin and/or BMP-2 were used to study the bone formation in a critical-sized bone defect created in a rat skull. Samples were retrieved for Micro-CT and histological analysis 12 weeks after surgery. The results indicated that BioCaP with or without the incorporation of Icariin had a positive effect on the osteogenic differentiation of MC3T3-E1. BioCaP with Icariin had better osteogenic efficiency, but had no influence on cell proliferation. BioCap + Icariin + BMP-2 showed better osteogenic potential compared with BioCaP with BMP-2 alone. The protein and mRNA expression of alkaline phosphatase and osteocalcin and mineralization were higher as well. In vivo, BioCaP incorporate internally with both Icariin and BMP-2 induced significantly more newly formed bone than the control group and BioCaP with either Icariin or BMP-2 did. Micro-CT analysis revealed that no significant differences were found between the bone mineral density induced by BioCaP with icariin and that induced by BioCaP with BMP-2. Therefore, co-administration of Icariin and BMP-2 was helpful for bone tissue engineering.

Introduction: Critical-sized defect (CSD) is one of the most challenging cases for orthopaedic surgeons. We aim to explore the therapeutic potential of the combination of bone marrow-derived mesenchymal stem cells (BM-MSCs), hydroxyapatite (HA) granules, bone morphogenetic protein-2 (BMP-2), and internal fixation for treating CSDs.Methods: This was a translational study performed during the period of January 2012 to 2016. Subjects were patients diagnosed with CSDs who had previously failed surgical attempts. They were treated with the combination of autologous BM-MSCs, HA granules, BMP-2, and mechanical stabilization. Post-operative pain level, functional outcome, defect volume, and radiological healing were evaluated after a minimum follow-up of 12 months.Results: A total of six subjects were recruited in this study. The pain was significantly reduced in all cases; with the decrease of mean preoperative visual analog scale (VAS) from 4 ± 2.2 to 0 after six month follow-up. Clinical functional outcome percentage increased significantly from 25 ± 13.7 to 70.79 ± 19.5. Radiological healing assessment using Tiedemann score also showed an increase from 0.16 ± 0.4 to 8 ± 3 at one year follow-up. No immunologic nor neoplastic side effects were found.Conclusions: The combination of autologous BM-MSCs, HA granules, and BMP-2 is safe and remains to be a good option for the definitive treatment for CSD with previous failed surgical attempts. Further studies with a larger sample size are required to be done. [ABSTRACT FROM AUTHOR]

• Critical-sized bone defect remains a challenge. • Many treatment options are available. • Tissue engineering is one feasible option. Introduction: Nonunion due to a critical-sized bone defect is a complicated problem. The healing process must fulfill three mandatory elements of osteogenesis, osteoinduction, and osteoconduction. One ideal source to provide an abundant number of osteogenic cells is from the process of the culture of bone marrow stem cells which demands the availability of processing facility. Unfortunately, this sophisticated option is not always feasible in every hospital in low-income to middle-income countries. We tried to fulfill the requirement of osteogenic cells by using simple and cost-effective bone marrow aspirate. We presented two cases of critical-sized diaphyseal bone defect treated with the combination of bone marrow aspirate, cancellous bone allograft, and platelet-rich plasma (PRP). Presentation of cases: The defect sizes were five and six centimeters in humerus and tibia respectively. We applied a combination of bone marrow aspirate, cancellous bone allograft, and PRP to promote bone healing in the defect sites. Both patients have achieved the good clinical and radiological outcome. Discussion: Critical-sized bone defects require the application of tissue engineering. Aspirated bone marrow can be used as a more affordable option to provide the element of osteogenic cells in bone healing. Combined with cancellous bone allograft and PRP, they fulfill the required ingredients to promote bone regeneration. Conclusion: Bone defects remain one of the most challenging conditions to treat in orthopedic. There are many options to treat the defect but the fundamental prerequisites of cells, scaffolds and growth factors for healing have developed into the concept of tissue engineering: osteogenesis, osteoinduction, and osteoconduction. [ABSTRACT FROM AUTHOR]

Icariin, a typical flavonol glycoside, is the main active component of Herba Epimedii, which was used to cure bone-related diseases in China for centuries. It has been reported that Icariin can be delivered locally by biomaterials and it has an osteogenic potential for bone tissue engineering. Biomimetic calcium phosphate (BioCaP) bone substitute is a novel drug delivery carrier system. Our study aimed to evaluate the osteogenic potential when Icariin was internally incorporated into the BioCaP granules. The BioCaP combined with Icariin and bone morphogenetic protein 2 (BMP-2) was investigated in vitro using an MC3T3-E1 cell line. We also investigated its efficacy to repair 8 mm diameter critical size bone defects in the skull of SD male rats. BioCaP was fabricated according to a well-established biomimetic mineralization process. In vitro , the effects of BioCaP alone or BioCaP with Icariin and/or BMP-2 on cell proliferation and osteogenic differentiation of MC3T3-E1 cells were systematically evaluated. In vivo , BioCaP alone or BioCaP with Icariin and/or BMP-2 were used to study the bone formation in a critical-sized bone defect created in a rat skull. Samples were retrieved for Micro-CT and histological analysis 12 weeks after surgery. The results indicated that BioCaP with or without the incorporation of Icariin had a positive effect on the osteogenic differentiation of MC3T3-E1. BioCaP with Icariin had better osteogenic efficiency, but had no influence on cell proliferation. BioCap + Icariin + BMP-2 showed better osteogenic potential compared with BioCaP with BMP-2 alone. The protein and mRNA expression of alkaline phosphatase and osteocalcin and mineralization were higher as well. In vivo , BioCaP incorporate internally with both Icariin and BMP-2 induced significantly more newly formed bone than the control group and BioCaP with either Icariin or BMP-2 did. Micro-CT analysis revealed that no significant differences were found between the bone mineral density induced by BioCaP with icariin and that induced by BioCaP with BMP-2. Therefore, co-administration of Icariin and BMP-2 was helpful for bone tissue engineering. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to assess the potential of poly (acrylic acid)/tricalcium phosphate nanoparticles (PAA/triCaPNPs) scaffold in terms of biocompatibility and osteoconductivity properties the in-vivo evaluation as well as to investigate the performance of PAA/triCaPNPs scaffold (with or without exosomes derived from UC-MSCs) for bone regeneration of rat critical-sized defect., Methods: PAA/triCaPNPs scaffold was made from acrylic acid (AA) monomer, N,N'-methylenebisacrylamide (MBAA), sodium bicarbonate (SBC), and ammonium persulfate (APS) through freeze-drying method. For in vivo evaluation, we randomly divided 24 rats into three groups. The rat calvarial bone defects were treated as follows: (1) Control group: defects without any treatment, (2) scaffold group: defects treated with scaffold only, (3) scaffold+exo group: defects treated with scaffold enriched with exosomes (1 μg/μL, 150 μg per rat). Eight- and 12-weeks post-surgery, half of the animals were sacrificed and bone regeneration was examined through micro-computerized tomography (µ-CT), histological staining, and immunohistochemistry (IHC)., Results: Quantitative analysis based on µ-CT scan images at 8 and 12 weeks post-implantation clearly indicated that healing rate for defects that were filled with scaffold enriched with exosome was significantly higher than defects filled with scaffold without exosome. The H&E and Masson staining results revealed that more new bone-like form developed in the scaffold+exo group than that in control and scaffold groups. Further, IHC staining for osteocalcin and CD31 confirmed that more bone healing in the scaffold+exo group at 12 weeks could be associated with osteogenesis and angiogenesis concurrently., Conclusion: In the present study, we aimed to investigate the therapeutic potential of PAA/triCaPNPs scaffold as a carrier of human UC-MSC-derived exosome to achieve the exosome-controlled release on calvarial bone defect. The in vivo results indicated that the exosome-enriched scaffold could effectively minify the defect area and improve the bone healing in rat model, and as such it could be an option for exosome-based therapy., Competing Interests: There is no competing financial interest., (© 2024 The Author(s).)

Background: Critical-sized bone defects are a significant challenge with limited effective reconstructive options. The Masquelet Technique (MT) offers a solution to help restore form and function. Although this technique has produced promising results; a clear mechanism has not been determined. Theories include that the induced membrane has osteogenic potential or the membrane acts as a physical barrier to prevent fibrous tissue ingrowth. We hypothesize the induced membrane acts primarily as a physical barrier and that a synthetic non-biological membrane will allow a comparable amount of bone volume in the defect site.Methods: Ten New Zealand rabbit forelimbs (n=10) were divided into three study groups. A critical sized defect of 3.5cm in the ulna was created. In the control group, a traditional MT was performed (n=4). The experimental arm varied by replacement of the PMMA with a non-porous (n=3) or porous (150um) (n=3) polytetrafluoroethylene (PTFE) membrane filled with allograft. Micro-CT analysis was done to compare bone volume to tissue volume ratios (BV/TV). Defect sections were examined histologically with alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) and von kossa (VK) staining.Results: MicroCT analysis comparing BV/TV between the control and experimental arms showed no difference. BV/TV of the MT was 7.77%±2.34 compared to porous 9.12%±3.66 and nonporous 9.76%±1.57 PTFE membranes (p1=0.761, p2=0.572, respectively). Histological sections from both samples stained for ALP and TRAP displayed osteoblastic and osteoclastic activity. There was a higher amount of ALP and TRAP positively stained cells near the native bone ends in comparison to the center of the defect, in both sample types.Conclusion and Significance: Replacing the induced membrane from the MT with a synthetic PTFE membrane illustrated that the membrane acts primarily as a functional barrier. Compared to the induced membrane, the PTFE membrane was able to display similar osteointegrative properties. These results allow for future optimization of the technique with the potential to further streamline towards a single stage procedure. [ABSTRACT FROM AUTHOR]

Background/Aims: Human dental pulp-derived mesenchymal stromal cells (hDPSCs) are promising seed cells for tissue engineering due to their easy accessibility and multilineage differentiation. Pannexin3 (Panx3) plays crucial roles during bone development and differentiation. The aim of the present study was to investigate the effect of Panx3 on osteogenesis of hDPSCs and the underlying mechanism. Methods: Utilizing qRT-PCR, Western blot, and immunohistochemistry, we explored the change of Panx3 during osteogenic differentiation of hDPSCs. Next, hDPSCs with loss (Panx3 knockdown) and gain (Panx3 overexpression) of Panx3 function were developed to investigate the effects of Panx3 on osteogenic differentiation of hDPSC and the underlying mechanism. Finally, a commercial β-TCP scaffold carrying Panx3-modified hDPSCs was utilized to evaluate bone defect repair. Results: Panx3 was upregulated during osteogenic differentiation in a time-dependent manner. Panx3 overexpression promoted osteogenic differentiation of hDPSCs, whereas depletion of Panx3 resulted in a decline of differentiation, evidenced by upregulated expression of mineralization-related markers, increased alkaline phosphatase (ALP) activity, and enhanced ALP and Alizarin red staining. Panx3 was found to interact with the Wnt/β-catenin signaling pathway, forming a negative feedback loop. However, Wnt/β-catenin did not contribute to enhancement of osteogenic differentiation as observed in Panx3 overexpression. Moreover, Panx3 promoted osteogenic differentiation of hDPSCs via increasing ERK signaling pathway. Micro-CT and histological staining results showed that Panx3-modified hDPSCs significantly improved ossification of critical-sized bone defects. Conclusion: These findings suggest that Panx3 is a crucial modulator of hDPSCs differentiation. [ABSTRACT FROM AUTHOR]

The scarcity of native periosteum poses a significant clinical barrier in the repair of critical-sized bone defects. The challenge of enhancing regenerative potential in bone healing is further compounded by oxidative stress at the fracture site. However, the introduction of artificial periosteum has demonstrated its ability to promote bone regeneration through the provision of appropriate mechanical support and controlled release of pro-osteogenic factors. In this study, a poly (l-lactic acid) (PLLA)/hyaluronic acid (HA)-based nanofibrous membrane was fabricated using the coaxial electrospinning technique. The incorporation of irisin into the core-shell structure of PLLA/HA nanofibers (PLLA/HA@Irisin) achieved its sustained release. In vitro experiments demonstrated that the PLLA/HA@Irisin membranes exhibited favorable biocompatibility. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) was improved by PLLA/HA@Irisin, as evidenced by a significant increase in alkaline phosphatase activity and matrix mineralization. Mechanistically, PLLA/HA@Irisin significantly enhanced the mitochondrial function of BMMSCs via the activation of the sirtuin 3 antioxidant pathway. To assess the therapeutic effectiveness, PLLA/HA@Irisin membranes were implanted in situ into critical-sized calvarial defects in rats. The results at 4 and 8 weeks post-surgery indicated that the implantation of PLLA/HA@Irisin exhibited superior efficacy in promoting vascularized bone formation, as demonstrated by the enhancement of bone matrix synthesis and the development of new blood vessels. The results of our study indicate that the electrospun PLLA/HA@Irisin nanofibers possess characteristics of a biomimetic periosteum, showing potential for effectively treating critical-sized bone defects by improving the mitochondrial function and maintaining redox homeostasis of BMMSCs., (© The Author(s) 2023. Published by Oxford University Press.)

Critical-sized bone defects resulting from trauma, inflammation, and tumor resections are individual in their size and shape. Implants for the treatment of such defects have to consider biomechanical and biomedical factors, as well as the individual conditions within the implantation site. In this context, 3D printing technologies offer new possibilities to design and produce patient-specific implants reflecting the outer shape and internal structure of the replaced bone tissue. The selection or modification of materials used in 3D printing enables the adaption of the implant, by enhancing the osteoinductive or biomechanical properties. In this study, scaffolds with bone spongiosa-inspired structure for extrusion-based 3D printing were generated. The computer aided design process resulted in an up scaled and simplified version of the bone spongiosa. To enhance the osteoinductive properties of the 3D printed construct, polycaprolactone (PCL) was combined with 20% (wt) calcium phosphate nano powder (CaP). The implants were designed in form of a ring structure and revealed an irregular and interconnected porous structure with a calculated porosity of 35.2% and a compression strength within the range of the natural cancellous bone. The implants were assessed in terms of biocompatibility and osteoinductivity using the osteosarcoma cell line MG63 and patient-derived mesenchymal stem cells in selected experiments. Cell growth and differentiation over 14 days were monitored using confocal laser scanning microscopy, scanning electron microscopy, deoxyribonucleic acid (DNA) quantification, gene expression analysis, and quantitative assessment of calcification. MG63 cells and human mesenchymal stem cells (hMSC) adhered to the printed implants and revealed a typical elongated morphology as indicated by microscopy. Using DNA quantification, no differences for PCL or PCL-CaP in the initial adhesion of MG63 cells were observed, while the PCL-based scaffolds favored cell proliferation in the early phases of culture up to 7 days. In contrast, on PCL-CaP, cell proliferation for MG63 cells was not evident, while data from PCR and the levels of calcification, or alkaline phosphatase activity, indicated osteogenic differentiation within the PCL-CaP constructs over time. For hMSC, the highest levels in the total calcium content were observed for the PCL-CaP constructs, thus underlining the osteoinductive properties., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Kühl, Gorb, Kern, Klüter, Kühl, Seekamp and Fuchs.)

Treating osteoporosis and associated bone fractures remains challenging for drug development in part due to potential off-target side effects and the requirement for long-term treatment. Here, we identify recombinant adeno-associated virus (rAAV)-mediated gene therapy as a complementary approach to existing osteoporosis therapies, offering long-lasting targeting of multiple targets and/or previously undruggable intracellular non-enzymatic targets. Treatment with a bone-targeted rAAV carrying artificial microRNAs (miRNAs) silenced the expression of WNT antagonists, schnurri-3 (SHN3), and sclerostin (SOST), and enhanced WNT/β-catenin signaling, osteoblast function, and bone formation. A single systemic administration of rAAVs effectively reversed bone loss in both postmenopausal and senile osteoporosis. Moreover, the healing of bone fracture and critical-sized bone defects was also markedly improved by systemic injection or transplantation of AAV-bound allograft bone to the osteotomy sites. Collectively, our data demonstrate the clinical potential of bone-specific gene silencers to treat skeletal disorders of low bone mass and impaired fracture repair., Competing Interests: Declaration of interests J.-H.S. is a scientific co-founder of AAVAA Therapeutics and holds equity in this company. G.G. is a scientific co-founder of AAVAA Therapeutics, Voyager Therapeutics, and Aspa Therapeutics and holds equity in these companies. G.G. is an inventor on patents with potential royalties licensed to Voyager Therapeutics, Aspa Therapeutics, and other biopharmaceutical companies. D.S.O. is a chief scientific officer of Osteogene Tech. These pose no conflicts for this study. The other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction Osteofibrous dysplasia is a rare non-neoplastic disease that is almost exclusive to pediatric tibial diaphysis. Wide excision of the lesion is recommended to avoid recurrence. However, such radical surgery will results in large segmental bone defects that will require further extensive reconstructive surgery. We report a novel approach of treating bone defect by implementing the diamond concept of bone healing using autologous bone marrow derived mesenchymal stem cells (BM-MSCs). Presentation of case An eight-year-old Indonesian male presented with severe bowing deformity of the left lower leg. Radiographic and histological analysis confirmed the diagnosis of osteofibrous dysplasia. A wide excision of the defect was made leaving a critical-sized bone defect. A combination of autologous transplantation of 50 million BM-MSCs, hydroxyapatite (HA) granules, bone morphogenic protein 2 (BMP-2) and Djoko-Zarov hybrid circular external fixator was used to treat the defect. The outcomes measured were subjective complaints, functionality based on LEFS and radiological assessments. Discussion Radiographic assessments showed successful new bone tissue formation and integration of implanted HA granules. The external fixator was removed at 42 weeks after adequate callus formation and clinical stability was achieved. The patient underwent progressive functional improvements and reached a near normal functionality of 90% LEFS at 84 week. No therapy side effect or complication was reported. Conclusion Osteofibrous dysplasia was successfully excised without signs of recurrence after 84-week follow-up. Autologous transplantation of augmented BM-MSCs has successfully created new normal bone tissue without causing any side effect and had significantly improved the patient’s quality of life. [ABSTRACT FROM AUTHOR]