Experimental Study on Rats with Critical-Size Bone Defects Comparing Effects of Autologous Bone Graft, Equine Bone Substitute Bio-Gen® Alone or in Association with Platelet-Rich Fibrin (PRF).

BACKGROUND: A critical-sized bone defect (CsBD) is considered one that will not heal spontaneously and requires reconstruction. This study aims to compare the results of using different bone reconstructive techniques and to study the potential of platelet-rich fibrin (PRF) to enhance the healing properties of a bone substitute (BS). METHODS: In this experimental study on rats, the treatment of critical-sized bone defects was carried out by analysing four groups: a control group in which the bone defect was left empty; a group treated with Bio-Gen®; another group in which the defect was treated with PRF in combination with Bio-Gen®; and the last that was treated with autologous bone graft (ABG). The defects were evaluated by microcomputed tomography (µCT) and then histomorphometrically. RESULTS: From both the histological and imagistic point of view, the best results were registered in the ABG group, followed by the group treated with Bio-Gen® with PRF, Bio-Gen® group, and control group, with statistically significant differences. CONCLUSIONS: A 5 mm defect in the rat radius can be considered critical. ABG showed the best results in treating the bone defect. PRF significantly enhanced the efficacy of Bio-Gen®.

Murine iPSC-Loaded Scaffold Grafts Improve Bone Regeneration in Critical-Size Bone Defects.

In certain situations, bones do not heal completely after fracturing. One of these situations is a critical-size bone defect where the bone cannot heal spontaneously. In such a case, complex fracture treatment over a long period of time is required, which carries a relevant risk of complications. The common methods used, such as autologous and allogeneic grafts, do not always lead to successful treatment results. Current approaches to increasing bone formation to bridge the gap include the application of stem cells on the fracture side. While most studies investigated the use of mesenchymal stromal cells, less evidence exists about induced pluripotent stem cells (iPSC). In this study, we investigated the potential of mouse iPSC-loaded scaffolds and decellularized scaffolds containing extracellular matrix from iPSCs for treating critical-size bone defects in a mouse model. In vitro differentiation followed by Alizarin Red staining and quantitative reverse transcription polymerase chain reaction confirmed the osteogenic differentiation potential of the iPSCs lines. Subsequently, an in vivo trial using a mouse model (n = 12) for critical-size bone defect was conducted, in which a PLGA/aCaP osteoconductive scaffold was transplanted into the bone defect for 9 weeks. Three groups (each n = 4) were defined as (1) osteoconductive scaffold only (control), (2) iPSC-derived extracellular matrix seeded on a scaffold and (3) iPSC seeded on a scaffold. Micro-CT and histological analysis show that iPSCs grafted onto an osteoconductive scaffold followed by induction of osteogenic differentiation resulted in significantly higher bone volume 9 weeks after implantation than an osteoconductive scaffold alone. Transplantation of iPSC-seeded PLGA/aCaP scaffolds may improve bone regeneration in critical-size bone defects in mice.

The Effect of Low-Intensity Pulsed Ultrasound on Bone Regeneration and the Expression of Osterix and Cyclooxygenase-2 during Critical-Size Bone Defect Repair.

Low-intensity pulsed ultrasound (LIPUS) is a form of ultrasound that utilizes low-intensity pulsed waves. Its effect on bones that heal by intramembranous ossification has not been sufficiently investigated. In this study, we examined LIPUS and the autologous bone, to determine their effect on the healing of the critical-size bone defect (CSBD) of the rat calvaria. The bone samples underwent histological, histomorphometric and immunohistochemical analyses. Both LIPUS and autologous bone promoted osteogenesis, leading to almost complete closure of the bone defect. On day 30, the bone volume was the highest in the autologous bone group (20.35%), followed by the LIPUS group (19.12%), and the lowest value was in the control group (5.11%). The autologous bone group exhibited the highest intensities of COX-2 (167.7 ± 1.1) and Osx (177.1 ± 0.9) expression on day 30. In the LIPUS group, the highest intensity of COX-2 expression was found on day 7 (169.7 ±1.6) and day 15 (92.7 ± 2.2), while the highest Osx expression was on day 7 (131.9 ± 0.9). In conclusion, this study suggests that LIPUS could represent a viable alternative to autologous bone grafts in repairing bone defects that are ossified by intramembranous ossification.

Corrective osteotomy of the distal radius with palmar locking plate osteosynthesis without bone grafting and without cortical contact.

The aim of this study was to assess bone healing and secondary fracture displacement after corrective osteotomy of the distal radius without any cortical contact using palmar locking plates without bone grafting. Between 2009 and 2021, 11 palmar corrective osteotomies of extra-articular malunited distal radius fractures and palmar plate fixations without the use of bone grafts and without cortical contact, were assessed. All patients showed complete osseous restoration and significant improvement in all radiographic parameters. Except for one patient, there were no secondary dislocations or loss of reduction in the postoperative follow-up. Bone grafts may not be mandatory for bone healing and prevention of secondary fracture displacement after palmar corrective osteotomy without cortical contact and fixation with palmar locking plate.Level of evidence: IV.

Experimental Early Stimulation of Bone Tissue Neo-Formation for Critical Size Elimination Defects in the Maxillofacial Region.

A biomaterial is proposed for closing extensive bone defects in the maxillofacial region. The composition of the biomaterial includes high-molecular chitosan, chondroitin sulfate, hyaluronate, heparin, alginate, and inorganic nanostructured hydroxyapatite. The purpose of this study is to demonstrate morphological and histological early signs of reconstruction of a bone cavity of critical size. The studies were carried out on 84 white female rats weighing 200-250 g. The study group consisted of 84 animals in total, 40 in the experimental group and 44 in the control group. In all animals, three-walled bone defects measuring 0.5 × 0.4 × 0.5 cm3 were applied subperiosteally in the region of the angle of the lower jaw and filled in the experimental group using lyophilized gel mass of chitosan-alginate-hydroxyapatite (CH-SA-HA). In control animals, the bone cavities were filled with their own blood clots after bone trepanation and bleeding. The periods for monitoring bone regeneration were 3, 5, and 7 days and 2, 3, 4, 6, 8, and 10 weeks. The control of bone regeneration was carried out using multiple morphological and histological analyses. Results showed that the following process is an obligatory process and is accompanied by the binding and release of angiogenic implantation: the chitosan construct actively replaced early-stage defects with the formation of full-fledged new bone tissue compared to the control group. By the 7th day, morphological analysis showed that the formation of spongy bone tissue could be seen. After 2 weeks, there was a pronounced increase in bone volume (p < 0.01), and at 6 weeks after surgical intervention, the closure of the defect was 70-80%; after 8 weeks, it was 100% without violation of bone morphology with a high degree of mineralization. Thus, the use of modified chitosan after filling eliminates bone defects of critical size in the maxillofacial region, revealing early signs of bone regeneration, and serves as a promising material in reconstructive dentistry.

Pretreatment of Mesenchymal Stem Cells with Electrical Stimulation as a Strategy to Improve Bone Tissue Engineering Outcomes.

Electrical stimulation (EStim), whether used alone or in combination with bone tissue engineering (BTE) approaches, has been shown to promote bone healing. In our previous in vitro studies, mesenchymal stem cells (MSCs) were exposed to EStim and a sustained, long-lasting increase in osteogenic activity was observed. Based on these findings, we hypothesized that pretreating MSC with EStim, in 2D or 3D cultures, before using them to treat large bone defects would improve BTE treatments. Critical size femur defects were created in 120 Sprague-Dawley rats and treated with scaffold granules seeded with MSCs that were pre-exposed or not (control group) to EStim 1 h/day for 7 days in 2D (MSCs alone) or 3D culture (MSCs + scaffolds). Bone healing was assessed at 1, 4, and 8 weeks post-surgery. In all groups, the percentage of new bone increased, while fibrous tissue and CD68+ cell count decreased over time. However, these and other healing features, like mineral density, bending stiffness, the amount of new bone and cartilage, and the gene expression of osteogenic markers, did not significantly differ between groups. Based on these findings, it appears that the bone healing environment could counteract the long-term, pro-osteogenic effects of EStim seen in our in vitro studies. Thus, EStim seems to be more effective when administered directly and continuously at the defect site during bone healing, as indicated by our previous studies.

Polylevolysine and Fibronectin-Loaded Nano-Hydroxyapatite/PGLA/Dextran-Based Scaffolds for Improving Bone Regeneration: A Histomorphometric in Animal Study.

The regeneration of large bone defects is still demanding, requiring biocompatible scaffolds, with osteoconductive and osteoinductive properties. This study aimed to assess the pre-clinical efficacy of a nano-hydroxyapatite (nano-HA)/PGLA/dextran-based scaffold loaded with Polylevolysine (PLL) and fibronectin (FN), intended for bone regeneration of a critical-size tibial defect, using an ovine model. After physicochemical characterization, the scaffolds were implanted in vivo, producing two monocortical defects on both tibiae of ten adult sheep, randomly divided into two groups to be euthanized at three and six months after surgery. The proximal left and right defects were filled, respectively, with the test scaffold (nano-HA/PGLA/dextran-based scaffold loaded with PLL and FN) and the control scaffold (nano-HA/PGLA/dextran-based scaffold not loaded with PLL and FN); the distal defects were considered negative control sites, not receiving any scaffold. Histological and histomorphometric analyses were performed to quantify the bone ingrowth and residual material 3 and 6 months after surgery. In both scaffolds, the morphological analyses, at the SEM, revealed the presence of submicrometric crystals on the surfaces and within the scaffolds, while optical microscopy showed a macroscopic 3D porous architecture. XRD confirmed the presence of nano-HA with a high level of crystallinity degree. At the histological and histomorphometric evaluation, new bone formation and residual biomaterial were detectable inside the defects 3 months after intervention, without differences between the scaffolds. At 6 months, the regenerated bone was significantly higher in the defects filled with the test scaffold (loaded with PLL and FN) than in those filled with the control scaffold, while the residual material was higher in correspondence to the control scaffold. Nano-HA/PGLA/dextran-based scaffolds loaded with PLL and FN appear promising in promoting bone regeneration in critical-size defects, showing balanced regenerative and resorbable properties to support new bone deposition.

3D-Printed Bioactive Scaffold Loaded with GW9508 Promotes Critical-Size Bone Defect Repair by Regulating Intracellular Metabolism.

The process of bone regeneration is complicated, and it is still a major clinical challenge to regenerate critical-size bone defects caused by severe trauma, infection, and tumor resection. Intracellular metabolism has been found to play an important role in the cell fate decision of skeletal progenitor cells. GW9508, a potent agonist of the free fatty acid receptors GPR40 and GPR120, appears to have a dual effect of inhibiting osteoclastogenesis and promoting osteogenesis by regulating intracellular metabolism. Hence, in this study, GW9508 was loaded on a scaffold based on biomimetic construction principles to facilitate the bone regeneration process. Through 3D printing and ion crosslinking, hybrid inorganic-organic implantation scaffolds were obtained after integrating 3D-printed ß-TCP/CaSiO3 scaffolds with a Col/Alg/HA hydrogel. The 3D-printed ß-TCP/CaSiO3 scaffolds had an interconnected porous structure that simulated the porous structure and mineral microenvironment of bone, and the hydrogel network shared similar physicochemical properties with the extracellular matrix. The final osteogenic complex was obtained after GW9508 was loaded into the hybrid inorganic-organic scaffold. To investigate the biological effects of the obtained osteogenic complex, in vitro studies and a rat cranial critical-size bone defect model were utilized. Metabolomics analysis was conducted to explore the preliminary mechanism. The results showed that 50 µM GW9508 facilitated osteogenic differentiation by upregulating osteogenic genes, including Alp, Runx2, Osterix, and Spp1 in vitro. The GW9508-loaded osteogenic complex enhanced osteogenic protein secretion and facilitated new bone formation in vivo. Finally, the results from metabolomics analysis suggested that GW9508 promoted stem cell differentiation and bone formation through multiple intracellular metabolism pathways, including purine and pyrimidine metabolism, amino acid metabolism, glutathione metabolism, and taurine and hypotaurine metabolism. This study provides a new approach to address the challenge of critical-size bone defects.

Bone regeneration of mouse critical-sized calvarial defects with human mesenchymal stem cell sheets co-expressing BMP2 and VEGF.

Background/purpose: Over-dependence on existing synthetic scaffolds and insufficient osteoinductive and vasculogenic growth factors have limited the development of bone regeneration. The study aimed to assess the feasibility of using marrow-derived mesenchymal stem cells (BMSCs) cell sheets co-expressing bone morphogenetic proteins 2 (BMP2) and vascular endothelial growth factor (VEGF) for repairing critical-sized calvarial defects. Materials and methods: BMSCs cell sheets were genetically engineered to express BMP2/VEGF alone or together. Alterations in osteogenic markers were examined by quantitative real-time PCR (qRT-PCR) and western blotting. A critical-sized calvarial bone defect model was used to investigate the osteogenesis effects of BMP2/VEGF cell sheets alone or in combination. The efficacy was assessed with micro-computed tomography (micro-CT) and histology. Results: In vitro, the expression of BMP2 and VEGF through lentiviral transduction was confirmed by qRT-PCR and western blotting against BMP2 and VEGF. Lentiviral delivery of BMP2 and VEGF resulted in the upregulation of osteogenic markers. In vivo, in a critical-sized calvarial bone defect model, 3D-reconstructed micro-CT images revealed that treatment of the calvarial defects with the BMP2/VEGF cell sheet resulted in significantly greater amounts of newly formed bone at 8 weeks after surgery than treatment with cell sheets with single gene transduction or vehicle controls. The results were confirmed by histological assessment by H&E staining and Masson staining. Conclusion: This study demonstrates that BMP2/VEGF co-expressing BMSCs sheets promote bone regeneration in critical-sized calvarial bone defects. The BMP2/VEGF cell sheets provide a functional bioactive scaffold for critical-size bone reconstruction.

SAXS imaging reveals optimized osseointegration properties of bioengineered oriented 3D-PLGA/aCaP scaffolds in a critical size bone defect model.

Healing large bone defects remains challenging in orthopedic surgery and is often associated with poor outcomes and complications. A major issue with bioengineered constructs is achieving a continuous interface between host bone and graft to enhance biological processes and mechanical stability. In this study, we have developed a new bioengineering strategy to produce oriented biocompatible 3D PLGA/aCaP nanocomposites with enhanced osseointegration. Decellularized scaffolds -containing only extracellular matrix- or scaffolds seeded with adipose-derived mesenchymal stromal cells were tested in a mouse model for critical size bone defects. In parallel to micro-CT analysis, SAXS tensor tomography and 2D scanning SAXS were employed to determine the 3D arrangement and nanostructure within the critical-sized bone. Both newly developed scaffold types, seeded with cells or decellularized, showed high osseointegration, higher bone quality, increased alignment of collagen fibers and optimal alignment and size of hydroxyapatite minerals.

Comparing the effect of demineralized versus hybrid dentin matrices on inducing bone regeneration in New Zealand white rabbits' Mandibular defect.

OBJECTIVES: The aim of this study was to compare the effect of using demineralized dentin matrix (DDM) versus hybrid dentin matrices; Demineralized and undemineralized dentin particles (DDM +UDDM) on inducing bone regeneration in mandibular defects. DESIGN: The study was conducted on fifty adult New Zealand rabbits, twenty for preparation of experimental materials and thirty for surgical procedures. They were randomly assigned into 3 equal groups as follow one control group: no treatment and two experimental groups including demineralized group: treated with DDM only; and hybrid group: treated with a hybrid of (50% DDM+50% UDD). A rounded critical size defect (10 mm in diameter- 5 mm in depth) was created in the body of mandible. After 3- and 6-weeks post-surgery, the bone regeneration was evaluated by light microscope, scanning electron microscope and histomorphometry. RESULTS: Histological, histomorphometrical observation and SEM revealed that both dentin matrices had largely resorbed and induced new bone formation at both experimental groups compared to the control group, with statistically higher percentage of new bone formation in the hybrid group. CONCLUSION: We concluded that although both dentin matrices induced new bone formation; however, hybrid dentin matrix yielded better results compared to DDM group.

Different Protocols of Combined Application of Photobiomodulation In Vitro and In Vivo Plus Adipose-Derived Stem Cells Improve the Healing of Bones in Critical Size Defects in Rat Models.

Introduction: Long bone segmental deficiencies are challenging complications to treat. Hereby, the effects of the scaffold derived from the human demineralized bone matrix (hDBMS) plus human adipose stem cells (hADSs) plus photobiomodulation (PBM) (in vitro and or in vivo) on the catabolic step of femoral bone repair in rats with critical size femoral defects (CDFDs) were evaluated with stereology and high stress load (HSL) assessment methods. Methods: hADSs were exposed to PBM in vitro; then, the mixed influences of hDBMS+hADS+PBM on CSFDs were evaluated. CSFDs were made on both femurs; then hDBMSs were engrafted into both CSFDs of all rats. There were 6 groups (G)s: G1 was the control; in G2 (hADS), hADSs only were engrafted into hDBMS of CSFD; in G3 (PBM) only PBM therapy for CSFD was provided; in G4 (hADS+PBM in vivo), seeded hADSs on hDBMS of CSFDs were radiated with a laser in vivo; in G5 (hADSs+PBM under in vitro condition), hADSs in a culture system were radiated with a laser, then transferred on hDBMS of CSFDs; and in G6 (hADS+PBM in conditions of in vivo and in vitro), laser-exposed hADSs were transplanted on hDBMS of CSFDs, and then CSFDs were exposed to a laser in vivo. Results: Groups 4, 5, and 6 meaningfully improved HSLs of CSFD in comparison with groups 3, 1, and 2 (all, P=0.001). HSL of G5 was significantly more than G4 and G6 (both, P=0.000). Gs 6 and 4 significantly increased new bone volumes of CSFD compared to Gs 2 (all, P=0.000) and 1 (P=0.001 & P=0.003 respectively). HSL of G 1 was significantly lower than G5 (P=0.026). Conclusion: HSLs of CSFD in rats that received treatments of hDBMS plus hADS plus PBM were significantly higher than treatments with hADS and PBM alone and control groups.

Differential dynamics of bone graft transplantation and mesenchymal stem cell therapy during bone defect healing in a murine critical size defect.

Background: A critical size bone defect is a clinical scenario in which bone is lost or excised due to trauma, infection, tumor, or other causes, and cannot completely heal spontaneously. The most common treatment for this condition is autologous bone grafting to the defect site. However, autologous bone graft is often insufficient in quantity or quality for transplantation to these large defects. Recently, tissue engineering methods using mesenchymal stem cells (MSCs) have been proposed as an alternative treatment. However, the underlying biological principles and optimal techniques for tissue regeneration of bone using stem cell therapy have not been completely elucidated. Methods: In this study, we compare the early cellular dynamics of healing between bone graft transplantation and MSC therapy in a murine chronic femoral critical-size bone defect. We employ high-dimensional mass cytometry to provide a comprehensive view of the differences in cell composition, stem cell functionality, and immunomodulatory activity between these two treatment methods one week after transplantation. Results: We reveal distinct cell compositions among tissues from bone defect sites compared with original bone graft, show active recruitment of MSCs to the bone defect sites, and demonstrate the phenotypic diversity of macrophages and T cells in each group that may affect the clinical outcome. Conclusion: Our results provide critical data and future directions on the use of MSCs for treating critical size defects to regenerate bone.Translational Potential of this article: This study showed systematic comparisons of the cellular and immunomodulatory profiles among different interventions to improve the healing of the critical-size bone defect. The results provided potential strategies for designing robust therapeutic interventions for the unmet clinical need of treating critical-size bone defects.

Self-Adhesive Hydrogel Biomimetic Periosteum to Promote Critical-Size Bone Defect Repair via Synergistic Osteogenesis and Angiogenesis.

The periosteum plays an important role in the regeneration of critical-size bone defects, with functions of recruiting multiple cells, accelerating vascular network reconstruction, and guiding bone tissue regeneration. However, these functions cannot be easily implemented by simply simulating the periosteum via a material structure design or by loading exogenous cytokines. Herein, inspired by the periosteal function, we propose a biomimetic periosteum preparation strategy to enhance natural polymer hydrogel membranes using inorganic bioactive materials. The biomimetic periosteum having bone tissue self-adhesive functions and resembling an extracellular matrix was prepared using dopamine-modified gelatin and oxidized hyaluronan (GA/HA), and micro/nanobioactive glass (MNBG) was further incorporated into the hydrogel to fabricate an organic/inorganic co-crosslinked hydrogel membrane (GA/HA-BG). The addition of MNBG enhanced the stability of the natural polymer hydrogel membrane, resulting in a sustained degradation time, biomineralization, and long-term release of ions. The Ca2+ and SiO44- ions released by bioactive glass were shown to recruit cells and promote the differentiation of bone marrow stromal cells into osteoblasts, initiating multicentric osteogenic behavior. Additionally, the bioactive ions were able to continuously stimulate the endogenous expression of vascular endothelial growth factor from human umbilical vein endothelial cells through the PI3K/Akt/HIF-1α pathway, which accelerated vascularization of the defect area and synergistically promoted the repair of bone defects. This organic-inorganic biomimetic periosteum has been proved to be effective and versatile in critical-size bone defect repair and is expected to provide a promising strategy for solving clinical issues.

A Retrospective Study on Mandibular Reconstruction Following Excision of Canine Acanthomatous Ameloblastoma.

The successful excision of a locally invasive tumor such as canine acanthomatous ameloblastoma (CAA) typically results in a mandibular contour-derforming, critical-size defect that alters the jaw kinematics, and may affect the patient's quality of life. In this case series, we describe our experience using the regenerative approach of a titanium locking plate and compression resistant matrix infused with rhBMP-2 for the immediate or delayed reconstruction following mandibulectomy for the excision of mandibular CAA in 11 dogs. Surgical planning included computed tomography (CT), with and without contrast, in all cases, and 3D-printed models in four cases. Tumor-free surgical margins were achieved in all dogs. Clinical and diagnostic imaging follow-up (mean, 23.1 months) were performed in-person (11 cases) and with CT/cone-beam computed tomography in most cases, with standard radiography (3 cases) and telemedicine being utilized in 5 cases. At 2 weeks postoperatively, hard tissue was palpable at the defect. Follow-up imaging at 1 month postoperatively revealed evidence of bridging new bone with a heterogeneous appearance, that remodeled over 3-6 months to bone of a similar size, shape and trabecular pattern as native bone. Histological evaluation of regenerated bone was available in two cases, and was supportive of our clinical and imaging findings of normal remodeled bone. Clinically, all dogs returned to a normal lifestyle, rapidly resumed eating and drinking, and exhibited normal occlusion. Complications included wound dehiscence in one dog and self-limiting exuberant bone formation in two dogs. Tumor regrowth, failure of the implant or fracture of the regenerated bone were not observed. We conclude that the mandibular reconstruction using a regenerative approach is safe, feasible, and results in restoration of mandibular contour in dogs following segmental and bilateral rostral mandibulectomy for benign but invasive oral tumors such as CAA.

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.

Novel Techniques and Future Perspective for Investigating Critical-Size Bone Defects.

A critical-size bone defect is a challenging clinical problem in which a gap between bone ends will not heal and will become a nonunion. The current treatment is to harvest and transplant an autologous bone graft to facilitate bone bridging. To develop less invasive but equally effective treatment options, one needs to first have a comprehensive understanding of the bone healing process. Therefore, it is imperative to leverage the most advanced technologies to elucidate the fundamental concepts of the bone healing process and develop innovative therapeutic strategies to bridge the nonunion gap. In this review, we first discuss the current animal models to study critical-size bone defects. Then, we focus on four novel analytic techniques and discuss their strengths and limitations. These four technologies are mass cytometry (CyTOF) for enhanced cellular analysis, imaging mass cytometry (IMC) for enhanced tissue special imaging, single-cell RNA sequencing (scRNA-seq) for detailed transcriptome analysis, and Luminex assays for comprehensive protein secretome analysis. With this new understanding of the healing of critical-size bone defects, novel methods of diagnosis and treatment will emerge.

Evaluation of the effects of preconditioned human stem cells plus a scaffold and photobiomodulation administration on stereological parameters and gene expression levels in a critical size bone defect in rats.

We assessed the impact of photobiomodulation (PBM) plus adipose-derived stem cells (ASCs) during the anabolic and catabolic stages of bone healing in a rat model of a critical size femoral defect (CSFD) that was filled with a decellularized bone matrix (DBM). Stereological analysis and gene expression levels of bone morphogenetic protein 4 (BMP4), Runt-related transcription factor 2 (RUNX2), and stromal cell-derived factor 1 (SDF1) were determined. There were six groups of rats. Group 1 was the untreated control or DBM. Study groups 2-6 were treated as follows: ASC (ASC transplanted into DBM, then implanted in the CSFD); PBM (CSFD treated with PBM); irradiated ASC (iASC) (ASCs preconditioned with PBM, then transplanted into DBM, and implanted in the CSFD); ASC + PBM (ASCs transplanted into DBM, then implanted in the CSFD, followed by PBM administration); and iASC + PBM (the same as iASC, except CSFDs were exposed to PBM). At the anabolic step, all treatment groups had significantly increased trabecular bone volume (TBV) (24.22%) and osteoblasts (83.2%) compared to the control group (all, p = .000). However, TBV in group iASC + PBM groups were superior to the other groups (97.48% for osteoblast and 58.8% for trabecular bone volume) (all, p = .000). The numbers of osteocytes in ASC (78.2%) and iASC + PBM (30%) groups were remarkably higher compared to group control (both, p = .000). There were significantly higher SDF (1.5-fold), RUNX2 (1.3-fold), and BMP4 (1.9-fold) mRNA levels in the iASC + PBM group compared to the control and some of the treatment groups. At the catabolic step of bone healing, TBV increased significantly in PBM (30.77%), ASC + PBM (32.27%), and iASC + PBM (35.93%) groups compared to the control group (all, p = .000). There were significantly more osteoblasts and osteocytes in ASC (71.7%, 62.02%) (p = .002, p = .000); PBM (82.54%, 156%), iASC (179%, 23%), and ASC + PBM (108%, 110%) (all, p = .000), and iASC + PBM (79%, 100.6%) (p = .001, p = .000) groups compared to control group. ASC preconditioned with PBM in vitro plus PBM in vivo significantly increased stereological parameters and SDF1, RUNX2, and BMP4 mRNA expressions during bone healing in a CSFD model in rats.

Metformin-Incorporated Gelatin/Hydroxyapatite Nanofiber Scaffold for Bone Regeneration.

Tissue engineering and regenerative medicine has gradually evolved as a promising therapeutic strategy to the modern health care of aging and diseased population. In this study, we developed a novel nanofibrous scaffold and verified its application in the critical bone defect regeneration. The metformin-incorporated nano-gelatin/hydroxyapatite fibers (NGF) was produced by electrospinning, cross-linked, and then characterized by X-ray powder diffractometer and Fourier-transform infrared spectroscopy. Cytotoxicity, cell adhesion, cell differentiation, and quantitative osteogenic gene and protein expression were analyzed by bone marrow stem cells (BMSCs) from rat. Rat forearm critical bone defect model was performed for the in vivo study. The NGF were characterized by their porous structures with proper interconnectivity without significant cytotoxic effects; the adhesion of BMSCs on the NGF could be enhanced. The osteogenic gene and protein expression were upregulated. Postimplantation, the new regenerated bone in bone defect was well demonstrated in the NGF samples. We demonstrated that the metformin-incorporated NGF greatly improved healing potential on the critical-size bone defect. Although metformin-incorporated NGF had advantageous effectiveness during bone regeneration, further validation is required before it can be applied to clinical applications. Impact statement Bone is the structure that supports the rest of the human body. Critical-size bone defect hinders the regeneration of damaged bone tissues and compromises the mechanical strength of the skeletal system. Characterized by their porous structures with proper interconnectivity, the electrospinning nano-gelatin/hydroxyapatite fibrous scaffold developed in this study can greatly improve the healing potential on the critical-size bone defect. Further validation can validate its potential clinical applications.

Osteoformation potential of an allogenic partially demineralized bone matrix in critical-size defects in the rat calvarium.

Allogenic demineralized bone matrix has been developed as a reliable alternative to the autologous bone graft. In the present study, we assessed the osteoformation potential of a partially demineralized bone matrix (PDBM) in a paste form obtained without an added carrier. This formulation included the preparation of cancelous bone from femoral heads after decellularision, delipidation, demineralization in HCl and autoclaving at 121 °C. Structural and biochemical characteristics of PDBM were determined using FTIR (Fourier transform infrared spectroscopy), hydroxyproline, DNA content assays, and optical ellipsometry. The osteoformation potential was evaluated in 8-, 6-, and 4-mm-diameter rat-calvarial bone defects by in vivo micro-CT analysis, performed immediately after surgery on days 0, 15, 30, 45, and 60. Moreover, histological and histomorphometric analyses were done on day 60. PDBM was compared to cancelous bone powder (BP) before its partial demineralization. The expression levels of selected inflammation-, angiogenesis-, and bone-related genes were also investigated by RT-PCR, 3, 7, and 14 days after surgery. Compared to the control group, the PDBM group exhibited a significant increase (p < 0.05) in radiopacity in 8-mm- and 6-mm-diameter defects at all time points tested. On day 60, the amount of newly-formed bone was greater (16 and 1.6 folds; p < 0.001; respectively) compared to that in control defects. No bone formation was observed in defects filled with BP regardeless of the size. In 8-mm-diameter defect, PDBM was effective enough to induce the upregulation of genes pertinent to inflammation (i.e., TNFα, IL-6, and IL-8), angiogenesis (i.e., VEGF, VWF), and osteogenesis (ALP, RUNX2, BGLAP, SP7) by day 3 after surgery. This study showed that the tested PDBM deeply influences the early critical events involved in bone regeneration and exhibits efficient osteoformation capacity, making it an attractive graft option for treating defects in periodontal and maxillofacial areas.