Critical-sized Femoral Defect Research Articles

Applying a trauma custom bridge system to reconstruct critical-sized femoral and tibial defects: Finite element analysis, osseointegration, and clinical outcomes.

Applying a trauma custom bridge system to reconstruct critical-sized femoral and tibial defects: Finite element analysis, osseointegration, and clinical outcomes.

Acceleration of bone repairation by BMSCs overexpressing NGF combined with NSA and allograft bone scaffolds

BackgroundRepairation of bone defects remains a major clinical problem. Constructing bone tissue engineering containing growth factors, stem cells, and material scaffolds to repair bone defects has recently become a hot research topic. Nerve growth factor (NGF) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but the low survival rate of the BMSCs during transplantation remains an unresolved issue. In this study, we investigated the therapeutic effect of BMSCs overexpression of NGF on bone defect by inhibiting pyroptosis.MethodsThe relationship between the low survival rate and pyroptosis of BMSCs overexpressing NGF in localized inflammation of fractures was explored by detecting pyroptosis protein levels. Then, the NGF+/BMSCs-NSA-Sca bone tissue engineering was constructed by seeding BMSCs overexpressing NGF on the allograft bone scaffold and adding the pyroptosis inhibitor necrosulfonamide(NSA). The femoral condylar defect model in the Sprague–Dawley (SD) rat was studied by micro-CT, histological, WB and PCR analyses in vitro and in vivo to evaluate the regenerative effect of bone repair.ResultsThe pyroptosis that occurs in BMSCs overexpressing NGF is associated with the nerve growth factor receptor (P75NTR) during osteogenic differentiation. Furthermore, NSA can block pyroptosis in BMSCs overexpression NGF. Notably, the analyses using the critical-size femoral condylar defect model indicated that the NGF+/BMSCs-NSA-Sca group inhibited pyroptosis significantly and had higher osteogenesis in defects.ConclusionNGF+/BMSCs-NSA had strong osteogenic properties in repairing bone defects. Moreover, NGF+/BMSCs-NSA-Sca mixture developed in this study opens new horizons for developing novel tissue engineering constructs.

Photobiomodulation and mesenchymal stem cell-conditioned medium for the repair of experimental critical-size defects.

Orthopedic surgeons face a significant challenge in treating critical-size femoral defects (CSFD) caused by osteoporosis (OP), trauma, infection, or bone tumor resections. In this study for the first time, the application of photobiomodulation (PBM) and bone marrow mesenchymal stem cell-conditioned medium (BM-MSC-CM) to improve the osteogenic characteristics of mineralized bone scaffold (MBS) in ovariectomy-induced osteoporotic (OVX) rats with a CSFD was tested. Five groups of OVX rats with CSFD were created: (1) Control (C); (2) MBS; (3) MBS + CM; (4) MBS + PBM; (5) MBS + CM + PBM. Computed tomography scans (CT scans), compression indentation tests, and histological and stereological analyses were carried out after euthanasia at 12 weeks following implantation surgery. The CT scan results showed that CSFD in the MBS + CM, MBS + PBM, and MBS + CM + PBM groups was significantly smaller compared to the control group (p = 0.01, p = 0.04, and p = 0.000, respectively). Moreover, the CSFD size was substantially smaller in the MBS + CM + PBM treatment group than in the MBS, MBS + CM, and MBS + PBM treatment groups (p = 0.004, p = 0.04, and p = 0.01, respectively). The MBS + PBM and MBS + CM + PBM treatments had significantly increased maximum force relative to the control group (p = 0.01 and p = 0.03, respectively). Bending stiffness significantly increased in MBS (p = 0.006), MBS + CM, MBS + PBM, and MBS + CM + PBM treatments (all p = 0.004) relative to the control group. All treatment groups had considerably higher new trabecular bone volume (NTBV) than the control group (all, p = 0.004). Combined therapies with MBS + PBM and MBS + CM + PBM substantially increased the NTBV relative to the MBS group (all, p = 0.004). The MBS + CM + PBM treatment had a markedly higher NTBV than the MBS + PBM (p = 0.006) and MBS + CM (p = 0.004) treatments. MBS + CM + PBM, MBS + PBM, and MBS + CM treatments significantly accelerated bone regeneration of CSFD in OVX rats. PBM + CM enhanced the osteogenesis of the MBS compared to other treatment groups.

A comparative in vitro and in vivo analysis of the impact of copper substitution on the cytocompatibility, osteogenic, and angiogenic properties of a borosilicate bioactive glass.

The 0106-B1-bioactive glass (BG) composition (in wt %: 37.5 SiO2, 22.6 CaO, 5.9 Na2O, 4.0 P2O5, 12.0 K2O, 5.5 MgO, and 12.5 B2O3) has demonstrated favorable processing properties and promising bone regeneration potential. The present study aimed to evaluate the biological effects of the incorporation of highly pro-angiogenic copper (Cu) in 0106-B1-BG in vitro using human bone marrow-derived mesenchymal stromal cells (BMSCs) as well as its in vivo potential for bone regeneration. CuO was added to 0106-B1-BG in exchange for CaO, resulting in Cu-doped BG compositions containing 1.0, 2.5 and 5.0 wt % CuO (composition in wt %: 37.5 SiO2, 21.6/ 20.1/17.6 CaO, 5.9 Na2O, 4.0 P2O5, 12.0 K2O, 5.5 MgO, 12.5 B2O3, and 1.0/ 2.5/ 5.0 CuO). In vitro, the BGs' impact on the viability, proliferation, and growth patterns of BMSCs was evaluated. Analyses of protein secretion, matrix formation, and gene expression were used for the assessment of the BGs' influence on BMSCs regarding osteogenic differentiation and angiogenic stimulation. The presence of Cu improved cytocompatibility, osteogenic differentiation, and angiogenic response when compared with unmodified 0106-B1-BG in vitro. In vivo, a critical-size femoral defect in rats was filled with scaffolds made from BGs. Bone regeneration was evaluated by micro-computed tomography. Histological analysis was performed to assess bone maturation and angiogenesis. In vivo effects regarding defect closure, presence of osteoclastic cells or vascular structures in the defect were not significantly changed by the addition of Cu compared with undoped 0106-B1-BG scaffolds. Hence, while the in vitro properties of the 0106-B1-BG were significantly improved by the incorporation of Cu, further evaluation of the BG composition is necessary to transfer these effects to an in vivo setting.

Strontium-loaded 3D intramedullary nail titanium implant for critical-sized femoral defect in rabbits.

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.

Bisphosphonates do not affect healing of a critical-size defect in estrogen-deficient mice

Bisphosphonates (BP) are anti-resorptive drugs that are widely used to prevent bone loss in osteoporosis. Since inhibition of bone resorption will cause a decrease in bone formation through a process called coupling, it is hypothesized that extended treatment protocols may impair bone healing. In this study, β-tri‑calcium-phosphate (βTCP) ceramics were inserted into critical-size long bone defects in estrogen-deficient mice under BP therapy. The study assessed the benefits of coating the ceramics with Bone Morphogenetic Protein-2 (BMP2) and an engineered BMP2 analogue (L51P) that inactivates BMP antagonists on the healing process, implant resorption, and bone formation.Female NMRI mice (11–12 weeks of age) were ovariectomized (OVX) or sham operated. Eight weeks later, after the manifestation of ovariectomy-induced osteoporotic bone changes, BP therapy with Alendronate (ALN) was commenced. After another five weeks, a femoral critical-size defect was generated, rigidly fixed, and βTCP-cylinders loaded with 0.25 μg or 2.5 μg BMP2, 2.5 μg L51P, and 0.25 μg BMP2/2.5 μg L51P, respectively, were inserted. Unloaded βTCP-cylinders were used as controls. Femora were collected six and twelve weeks post-implantation.Histological and micro-computer tomography (MicroCT) evaluation revealed that insertion of cylinders coated with 2.5 μg BMP2 accelerated fracture repair and induced significant bone formation compared to controls (unloaded cylinders or coated with 2.5 μg L51P, 0.25 μg BMP2) already six weeks post-implantation, independent of estrogen-deficiency and BP therapy. The simultaneous administration of BMP2 and L51P (0.25 μg BMP2/2.5 μg L51P) did not promote fracture healing six and twelve weeks post-implantation. Moreover, new bone formation within the critical-size defect was directly linked to the removal of the βTCP-implant in all experimental groups. No evidence was found that long-term therapy with ALN impaired the resorption of the implanted graft. However, osteoclast transcriptome signature was elevated in sham and OVX animals upon treatment with BP, with transcript levels being higher at six weeks than at twelve weeks post-surgery. Furthermore, the transcriptome profile of the developing repair tissue confirmed an accelerated repair process in animals treated with 2.5 μg BMP2 implants. L51P did not increase the bioefficacy of BMP2 in the applied defect model.The present study provides evidence that continuous administration of BP does not inhibit implant resorption and does not alter the kinetics of the healing process of critical-size long bone defects. Furthermore, the BMP2 variant L51P did not enhance the bioefficacy of BMP2 when applied simultaneously to the femoral critical-size defect in sham and OVX mice.

Therapeutic Effects of a Placental Tissue-Derived Allograft on Fracture Healing

Background:As 5-10% of fractures will not heal without medical intervention, there is an ongoing need for effective treatments to promote fracture healing. CTM Biomedical produces human placental tissue-derived allografts that are used clinically and may assist in healing; however, no pre-clinical studies assessing these products have been performed. Our study investigating the impact of CTM products on the healing of a standard femoral fracture and a critical sized femoral defect (CSD) aims to fill this gap. We hypothesize that CTM product application will improve fracture healing and reduce pain-related behaviors.
 Methods:Femoral fractures were induced in 45 mice. CTM membrane, CTM paste, a combination of CTM membrane and paste, or saline was applied to each fracture. X-rays were taken twice weekly over 22 days, and blinded modified Radiological Union Scale for Tibia (mRUST) fracture scoring was performed. Complete blood analysis was conducted weekly. Following euthanasia 23 days post-surgery, μCT and histomorphometric analyses were conducted. CSDs have also been surgically induced in the femurs of 95 mice, with plans for similar fracture analyses.
 Results:CTM product application did not significantly alter the levels of inflammatory cells, suggesting that the mice did not undergo immunological reactions. mRUST scoring indicated that CTM products may not alter fracture healing rates. However, combined application of CTM membrane and paste significantly increased the fracture callus’s mineralized volume (by ~90%) and the percent of the callus that was bone. CTM membrane and paste application also led to an increased threshold for hind paw withdrawal, suggesting that CTM products may decrease pain-related behaviors.
 Future Directions:We hypothesize that in our CSD model, mice treated with the combination of CTM membrane and paste will display improved fracture healing and decreased pain-related behaviors. If shown to be effective, CTM product use may decrease fracture nonunion risk and increase comfort.

Rehabilitation exercise-driven symbiotic electrical stimulation system accelerating bone regeneration.

Electrical stimulation can effectively accelerate bone healing. However, the substantial size and weight of electrical stimulation devices result in reduced patient benefits and compliance. It remains a challenge to establish a flexible and lightweight implantable microelectronic stimulator for bone regeneration. Here, we use self-powered technology to develop an electric pulse stimulator without circuits and batteries, which removes the problems of weight, volume, and necessary rigid packaging. The fully implantable bone defect electrical stimulation (BD-ES) system combines a hybrid tribo/piezoelectric nanogenerator to provide biphasic electric pulses in response to rehabilitation exercise with a conductive bioactive hydrogel. BD-ES can enhance multiple osteogenesis-related biological processes, including calcium ion import and osteogenic differentiation. In a rat model of critical-sized femoral defects, the bone defect was reversed by electrical stimulation therapy with BD-ES and subsequent bone mineralization, and the femur completely healed within 6 weeks. This work is expected to advance the development of symbiotic electrical stimulation therapy devices without batteries and circuits.

THE IMPACT OF METABOLISM DYSREGULATION IN SCAFFOLD-GUIDED LARGE-VOLUME BONE REGENERATION

The ability of the body to constantly maintain metabolism homeostasis while fulling the heightened energy and macromolecule demand is crucial to ensure successful tissue healing outcomes. Studies investigating the local metabolic environment during healing are scarce to date. Here, using Type 2 Diabetes (T2D) as a study model, we investigate the impact of metabolism dysregulation on scaffold-guided large-volume bone regeneration. Our study treated wild-type or T2D rats with 5 mm critical-sized femoral defects with 3D-printed polycaprolactone (PCL) scaffolds with 70% porosity. Metabolomics was leveraged for a holistic view of metabolism alteration as healing progress and correlated to regenerated bone tissue volume and quality assessed using micro-computed tomography (µ-CT), histology, and immunohistology. Semi-targeted metabolomics analysis indicated dysregulation in the glycolysis and TCA cycle – the main energy production pathways, in T2D compared to healthy animals. The abundance of metabolites substrates, i.e., amino acids – for protein/ extracellular matrix synthesis was also affected in T2D. Tissue-level metabolites observations aligned with morphological observation with less newly formed bone observed in T2D than wild-type rats. This study enlightens the metabolism landscape during scaffold-guided large-volume bone regeneration in wild-type vs. T2D to further guide the personalization of the scaffold to drive successful regeneration.

A NOVEL SELECTIVE PROSTAGLANDIN-RECEPTOR EP4-AGONIST PROMOTES SPINAL FUSION IN RATS

A novel EP4 selective agonist (KMN-159) was developed [1] and has been proven that it can act as an osteopromotive factor to repair critical-size femoral bone defects in rats at a dose-dependent manner [2]. Based on its osteopromotive properties, we hypothesized that KMN-159 could also aid in bone formation for spinal fusion. Therefore, the aim of this study was to investigate its spinal fusion effect in a dorsolateral spinal fusion model in rats. This study was performed on 192, 10-week-old male Wistar rats. The rats were randomized into 8 groups (n = 12 per group): 1) SHAM (negative control), 2) MCM (scaffold only), 3) MCM + 20 µg BMP-2 (positive control), 4-8) MCM + 0.2, 2, 20, 200 or 2000 µg KMN-159. A posterolateral intertransverse process spinal fusion at L4 to L5 was performed bilaterally by implanting group dependent scaffolds (see above) or left empty in the SHAM group (protocol no. 25-5131/474/38). Animals were euthanized after 3 weeks and 6 weeks for µCT and biomechanical testing analysis. The results showed that KMN-159 promoted new bone formation in a dose-dependent manner at 3 weeks and 6 weeks as verified by µCT. The biomechanical testing showed that the dose of 20, 200 and 2000 µg KMN-159 groups obtained comparable strength with BMP-2 group, which higher than SHAM, MCM and lower doses of 0.2 and 2 µg KMN-159 groups. In conclusion, KMN-159 could be a potential replacement of BMP-2 as a novel osteopromotive factor for spinal fusion.Acknowledgements: We are grateful to Ulrike Heide, Anna-Maria Placht (assistance with surgeries) as well as Suzanne Manthey & Annett Wenke (histology).

CALCIUM SULFATE/HYDROXYAPATITE-MEDIATED CONTROLLED CO-DELIVERY OF BMP-2 AND ZOLEDRONIC ACID ENHANCES SPINAL FUSION

Although bone morphogenetic protein 2 (BMP-2) has been FDA-approved for spinal fusion for decades, its disadvantages of promoting osteoclast-based bone resorption and suboptimal carrier (absorbable collagen sponge) leading to premature release of the protein limit its clinical applications. Our recent study showed an excellent effect on bone regeneration when BMP-2 and zoledronic acid (ZA) were co-delivered based on a calcium sulphate/hydroxyapatite (CaS/HA) scaffold in a rat critical-size femoral defect model. Therefore, the aim of this study was to evaluate whether local application of BMP-2 and ZA released from a CaS/HA scaffold is favorable for spinal fusion. We hypothesized that CaS/HA mediated controlled co-delivery of rhBMP-2 and ZA could show an improved effect in spinal fusion over BMP-2 alone. 120, 8-week-old male Wistar rats (protocol no. 25-5131/474/38) were randomly divided into six groups in this study (CaS/HA, CaS/HA + BMP-2, CaS/HA + systemic ZA, CaS/HA + local ZA, CaS/HA + BMP-2 + systemic ZA, CaS/HA + BMP-2 + local ZA). A posterolateral spinal fusion at L4 to L5 was performed bilaterally by implanting group-dependent scaffolds. At 3 weeks and 6 weeks, 10 animals per group were euthanized for µCT, histological staining, or mechanical testing. µCT and histological results showed that the CaS/HA + BMP-2 + local ZA group significantly promoted bone regeneration than other treated groups. Biomechanical testing showed breaking force in CaS/HA + BMP + local ZA group was significantly higher than other groups at 6 weeks. In conclusion, the CaS/HA-based biomaterial functionalized with bioactive molecules rhBMP-2 and ZA enhanced bone formation and concomitant spinal fusion outcomeAcknowledgements: Many thanks to Ulrike Heide, Anna-Maria Placht (assistance with surgeries) as well as Suzanne Manthey & Annett Wenke (histology).

Biological effects of a zinc-substituted borosilicate bioactive glass on human bone marrow derived stromal cells in vitro and in a critical-size femoral defect model in rats in vivo.

The borosilicate 0106-B1-bioactive glass (BG) composition (in wt%: 37.5 SiO2, 22.6 CaO, 5.9 Na2O, 4.0P2O5, 12.0 K2O, 5.5 MgO, 12.5 B2O3) has shown favorable processing characteristics and bone regeneration ability. This study investigated the addition of zinc (Zn) to 0106-B1-BG as an approach to improve this BG's biological properties. Different proportions of ZnO were substituted for CaO in 0106-B1-BG, resulting in three new BG-compositions: 1-Zn-BG, 2-Zn-BG, 3-Zn-BG (in wt%: 37.5 SiO2, 21.6/20.1/17.6 CaO, 4.0 P2O5, 5.9 Na2O, 12.0 K2O, 5.5 MgO, 12.5 B2O3 and 1.0/2.5/5.0 ZnO). Effects of the BG compositions on cytocompatibility, osteogenic differentiation, extracellular matrix deposition, and angiogenic response of human bone marrow-derived mesenchymal stromal cells (BMSCs) were evaluated in vitro. Angiogenic effects were assessed using a tube formation assay containing human umbilical vein endothelial cells. The in vivo osteogenic and angiogenic potentials of 3-Zn-BG were investigated in comparison to the Zn-free 0106-B1-BG in a rodent critical-size femoral defect model. The osteogenic differentiation of BMSCs improved in the presence of Zn. 3-Zn-BG showed enhanced angiogenic potential, as confirmed by the tube formation assay. While Zn-doped BGs showed clearly superior biological properties in vitro, 3-Zn-BG and 0106-B1-BG equally promoted the formation of new bone in vivo; however, 3-Zn-BG reduced osteoclastic cells and vascular structures in vivo. The acquired data suggests that the differences regarding the in vivo and in vitro results may be due to modulation of inflammatory responses by Zn, as described in the literature. The inflammatory effect should be investigated further to promote clinical applications of Zn-doped BGs.

Development of artificial bone graft via in vitro endochondral ossification (ECO) strategy for bone repair

Endochondral ossification (ECO) is a form of bone formation whereby the newly deposited bone replaces the cartilage template. A decellularized artificial cartilage graft (dLhCG), which is composed of hyaline cartilage matrixes, has been developed in our previous study. Herein, the osteogenesis of bone marrow-derived MSCs in the dLhCG through chondrogenic differentiation, chondrocyte hypertrophy, and subsequent transdifferentiation induction has been investigated by simulating the physiological processes of ECO for repairing critical-sized bone defects. The MSCs were recellularized into dLhCGs and subsequently allowed to undergo a 14-day proliferation period (mrLhCG). Following this, the mrLhCG constructs were subjected to two distinct differentiation induction protocols to achieve osteogenic differentiation: chondrogenic medium followed by chondrocytes culture medium with a high concentration of fetal bovine serum (CGCC group) and canonical osteogenesis inducing medium (OI group). The formation of a newly developed artificial bone graft, ossified dLhCG (OsLhCG), as well as its capability of aiding bone defect reconstruction were characterized by in vitro and in vivo trials, such as mRNA sequencing, quantitative real-time PCR (qPCR), immunohistochemistry, the greater omentum implantation in nude mice, and repair for the critical-sized femoral defects in rats. The results reveal that the differentiation induction of MSCs in the CGCC group can realize in vitro ECO through chondrogenic differentiation, hypertrophy, and transdifferentiation, while the MSCs in the OI group, as expected, realize ossification through direct osteogenic differentiation. The angiogenesis and osteogenesis of OsLhCG were proved by being implanted into the greater omentum of nude mice. Besides, the OsLhCG exhibits the capability to achieve the repair of critical-size femoral defects.

An engineered periosteum for efficient delivery of rhBMP-2 and mesenchymal progenitor cells during bone regeneration

During bone regeneration, the periosteum acts as a carrier for key regenerative cues, delivering osteochondroprogenitor cells and crucial growth factors to the injured bone. We developed a biocompatible, 3D polycaprolactone (PCL) melt electro-written membrane to act as a mimetic periosteum. Poly (ethyl acrylate) coating of the PCL membrane allowed functionalization, mediated by fibronectin and low dose recombinant human BMP-2 (rhBMP-2) (10-25 μg/ml), resulting in efficient, sustained osteoinduction in vitro. In vivo, rhBMP-2 functionalized mimetic periosteum demonstrated regenerative potential in the treatment of rat critical-size femoral defects with highly efficient healing and functional recovery (80%-93%). Mimetic periosteum has also proven to be efficient for cell delivery, as observed through the migration of transplanted periosteum-derived mesenchymal cells to the bone defect and their survival. Ultimately, mimetic periosteum demonstrated its ability to deliver key stem cells and morphogens to an injured site, exposing a therapeutic and translational potential in vivo when combined with unprecedentedly low rhBMP-2 doses.

Engineering of a NIR-activable hydrogel-coated mesoporous bioactive glass scaffold with dual-mode parathyroid hormone derivative release property for angiogenesis and bone regeneration.

Osteogenesis, osteoclastogenesis, and angiogenesis play crucial roles in bone regeneration. Parathyroid hormone (PTH), an FDA-approved drug with pro-osteogenic, pro-osteoclastogenic and proangiogenic capabilities, has been employed for clinical osteoporosis treatment through systemic intermittent administration. However, the successful application of PTH for local bone defect repair generally requires the incorporation and delivery by appropriate carriers. Though several scaffolds have been developed to deliver PTH, they suffer from the weaknesses such as uncontrollable PTH release, insufficient porous structure and low mechanical strength. Herein, a novel kind of NIR-activable scaffold (CBP/MBGS/PTHrP-2) with dual-mode PTHrP-2 (a PTH derivative) release capability is developed to synergistically promote osteogenesis and angiogenesis for high-efficacy bone regeneration, which is fabricated by integrating the PTHrP-2-loaded hierarchically mesoporous bioactive glass (MBG) into the N-hydroxymethylacrylamide-modified, photothermal agent-doped, poly(N-isopropylacrylamide)-based thermosensitive hydrogels through assembly process. Upon on/off NIR irradiation, the thermoresponsive hydrogel gating undergoes a reversible phase transition to allow the precise control of on-demand pulsatile and long-term slow release of PTHrP-2 from MBG mesopores. Such NIR-activated dual-mode delivery of PTHrP-2 by this scaffold enables a well-maintained PTHrP-2 concentration at the bone defect sites to continually stimulate vascularization and promote osteoblasts to facilitate and accelerate bone remodeling. In vivo experiments confirm the significant improvement of bone reparative effect on critical-size femoral defects of rats. This work paves an avenue for the development of novel dual-mode delivery systems for effective bone regeneration.

HISTOLOGICAL EVALUATION OF REPARATIVE OSTEOGENESIS IN CRITICAL SIZE FEMORAL BONE DEFECTS IN RATS OF DIFFERENT AGES AFTER INTRODUCTION OF ALLOGRAFTS SATURATED WITH BLOOD PLASMA GROWTH FACTORS

The increase in injuries and gunshot wounds because of the war in Ukraine makes it imperative to find methods for optimizing bone regeneration and filling large-size bone defects. Aim. Study morphological features of reparative osteogenesis when critical size femoral bone defects in rats in the early reproductive and mature stages are filled with allografts saturated with blood plasma growth factors (GF). Methods. Defects (3 × 3 mm) were created in the distal femoral metaphysis of 60 white laboratory rats, 3-months-old (n = 30) and 12-months-old (n = 30). The defects were filled with bone allografts saturated with GF in the two experimental groups (AlloG+GF), and unsaturated bone allografts in the two control groups (AlloG). All groups contained 15 rats of each age. At 14, 28 and 90 days after the surgery, 5 rats from each group were sacrificed, and histological analyses were performed. Results. In the AlloGgroups, excessive formation of connective tissue was observed 14 and 28 days after the surgery, most evident in the 3-monthold rats. In the AlloG+GF groups, bone formation was delayed at 14 days independent of age, while at 28 and 90 days, the area of bone trabeculae did not differ from the values in the AlloG groups. Throughout the experiment, decreases in allograft area (almost all of it was replaced by bone after 90 days) and connective tissue (completely absent in 3-month-old rats after 90 days) were observed in both AlloG+GF groups. The area of bone trabeculae increased in the period from 14 to 28 days. Conclusion. Saturating allografts with blood plasma growth factors facilitates an increase in the rate at which allografts are replaced by bone tissue, independent of the recipient’s age. However, excessive formation of connective tissues in the defect 14 and 28 days after the surgery, especiallyin 3-month-old rats, may negatively affect the mechanical properties of the bone, which should be considered in clinical practice.

A comparative in vitro and in vivo analysis of the biological properties of the 45S5-, 1393-, and 0106-B1-bioactive glass compositions using human bone marrow-derived stromal cells and a rodent critical size femoral defect model

Since the introduction of the 45S5-bioactive glass (BG), numerous new BG compositions have been developed. Compared to the 45S5-BG, 1393-BG shows favorable processing properties due to its low crystallization tendency and the 1393-BG-based borosilicate 0106-B1-BG exhibits improved angiogenic properties due to its boron content. Despite their close (chemical) relationship, the biological properties of the mentioned BG composition have not yet been comparatively examined. In this study, the effects of the BGs on proliferation, viability, osteogenic differentiation, and angiogenic factor production of human bone marrow-derived mesenchymal stromal cells were assessed. Scaffolds made of the BGs were introduced in a critical-sized femur defect model in rats in order to analyze their impact on bone defect regeneration. In vitro, 1393-BG and 0106-B1-BG outperformed 45S5-BG with regard to cell proliferation and viability. 1393-BG enhanced osteogenic differentiation; 0106-B1-BG promoted angiogenic factor production. In vivo, 0106-B1-BG and 45S5-BG outperformed 1393-BG in terms of angiogenic and osteoclastic response resulting in improved bone regeneration. In conclusion, the biological properties of BGs can be significantly modified by tuning their composition. Demonstrating favorable processing properties and an equally strong in vivo bone regeneration potential as 45S5-BG, 0106-B1-BG qualifies as a basis to incorporate other bioactive ions to improve its biological properties.

The enhancer landscape predetermines the skeletal regeneration capacity of stromal cells.

Multipotent stromal cells are considered attractive sources for cell therapy and tissue engineering. Despite numerous experimental and clinical studies, broad application of stromal cell therapeutics is not yet emerging. A major challenge is the functional diversity of available cell sources. Here, we investigated the regenerative potential of clinically relevant human stromal cells from bone marrow (BMSCs), white adipose tissue, and umbilical cord compared with mature chondrocytes and skin fibroblasts in vitro and in vivo. Although all stromal cell types could express transcription factors related to endochondral ossification, only BMSCs formed cartilage discs in vitro that fully regenerated critical-size femoral defects after transplantation into mice. We identified cell type-specific epigenetic landscapes as the underlying molecular mechanism controlling transcriptional stromal differentiation networks. Binding sites of commonly expressed transcription factors in the enhancer and promoter regions of ossification-related genes, including Runt and bZIP families, were accessible only in BMSCs but not in extraskeletal stromal cells. This suggests an epigenetically predetermined differentiation potential depending on cell origin that allows common transcription factors to trigger distinct organ-specific transcriptional programs, facilitating forward selection of regeneration-competent cell sources. Last, we demonstrate that viable human BMSCs initiated defect healing through the secretion of osteopontin and contributed to transient mineralized bone hard callus formation after transplantation into immunodeficient mice, which was eventually replaced by murine recipient bone during final tissue remodeling.

Microfibril-Associated Glycoprotein-2 Promoted Fracture Healing via Integrin αvβ3/PTK2/AKT Signaling

Fracture healing is a complex physiological process in which angiogenesis plays an essential role. Microfibril-associated glycoprotein-2 (MAGP2) has been reported to possess a proangiogenic activity via integrin αvβ3, yet its role in bone repair is unexplored. In this study, a critical-sized femoral defect (2 mm) was created in mice, followed by the delivery of an adenovirus-based MAGP2 overexpression vector or its negative control at the fracture site. At days 7, 14, 21, and 28 postfracture, bone fracture healing was evaluated by radiography, micro-computed tomography, and histopathologic analysis. Adenovirus-based MAGP2 overexpression vector–treated mice exhibited increased bone mineral density and bone volume fraction. MAGP2 overexpression contributed to an advanced stage of endochondral ossification and induced cartilage callus into the bony callus. Further analysis indicated that MAGP2 was associated with enhanced angiogenesis, as evidenced by marked MAGP2 and integrin αvβ3 costaining and increased endothelial cell markers such as endomucin and CD31 levls, as well as elevated phosphorylation of protein tyrosine kinase 2 (PTK2) and AKT serine/threonine kinase 1 (AKT) in the callus. In vitro, recombinant human MAGP2 treatment enhanced the viability, migration, and tube formation ability of human microvascular endothelial cells, which was partially reversed by integrin αvβ3 inhibition or MK-2206, a specific AKT inhibitor. Inhibition of integrin αvβ3 abolished MAGP2-induced PTK2 and AKT activation. Taken together, our data provide the first evidence that MAGP2 promotes angiogenesis and bone formation by activating the integrin αvβ3/PTK2/AKT signaling pathway.

Bone Healing of Critical-Sized Femoral Defects in Rats Treated with Erythropoietin Alone or in Combination with Xenograft.

Critical-size bone defect models are the standard in studies of the osteogenic potential of biomaterials. The present investigation aimed to evaluate the ability of recombinant human erythropoietin (EPO) to induce trabecular bone healing either alone or combined with a xenograft in a rat femoral critical-size defect model. Five-mm bone defects were created in the femoral diaphysis of fifty-six skeletally mature male Wistar albino rats. The animals were divided into six groups: one control group and five experimental groups. The defects in the control group were left empty, whereas an absorbable collagen cone soaked either with saline or erythropoietin (alone or in combination with xenograft) was placed in locally treated groups. The systemic treatment group received EPO subcutaneously. Bone formation was objectively evaluated through radiography, osteodensitometry and histological examination on post-operative days 30 and 90. The results demonstrate that EPO, locally applied on a collagen scaffold, was capable of inducing bone healing, whereas the single systemically administered high EPO dose had only an insignificant effect on bone formation. The combination of EPO with a bone substitute under the form of cancellous granules resulted in more rapid integration between the xenograft and host bone.