A novel configuration for the fixation of intra-articular C2.3 distal humerus fractures with the potential for minimally invasive surgery: a biomechanical evaluation and finite element analysis.

BACKGROUND: Distal humerus fractures are a challenge to treat, and the current standard of care, open reduction internal fixation with a double-plate, has a high rate of complications. We proposed a novel internal fixation configuration, lateral intramedullary nail and medial plate (LINMP) and verified its rigidity through biomechanical tests and finite element analysis. METHODS: The study involved biomechanical testing of 30 synthetic humerus models to compare 2 different fixation systems for an AO 13C-2.3 type fracture. The orthogonal double-plate (ODP) group and the LINMP group were compared through biomechanical testing to measure stiffness and failure load fewer than 3 working conditions. Based on the results, we optimized the intramedullary nail by eliminating the holes at the distal end of the nail and incorporating a 2-hole external locking plate. The Finite element analysis was also conducted to further compare the modified LINMP configuration with the previous 2 fixation configurations. RESULTS: In biomechanical tests, the ODP group exhibited lower stiffness under bending and compression forces compared to the LINMP group, but higher stiffness and failure loads under torsion force. In finite element analysis, the modified LINMP reduces the maximum stress of the fixation structure without significantly reducing the stiffness under bending stress and axial compression conditions. In torsion stress conditions, the modified LINMP enhances both the maximum stress and the stiffness, although it remains marginally inferior to the ODP structure. CONCLUSION: Our study demonstrates that the innovative LINMP presents comparable or slightly superior concerning bending and axial loading compared to orthogonal double-plate osteosynthesis for distal humeral intra-articular fractures, which might become a minimally invasive option for these fractures.

Combined Molybdenum Gelatine Methacrylate Injectable Nano-Hydrogel Effective Against Diabetic Bone Regeneration.

Introduction: Bone defects in diabetes mellitus (DM) remain a major challenge for clinical treatment. Fluctuating glucose levels in DM patients lead to excessive production of reactive oxygen species (ROS), which disrupt bone repair homeostasis. Bone filler materials have been widely used in the clinical treatment of DM-related bone defects, but overall they lack efficacy in improving the bone microenvironment and inducing osteogenesis. We utilized a gelatine methacrylate (GelMA) hydrogel with excellent biological properties in combination with molybdenum (Mo)-based polyoxometalate nanoclusters (POM) to scavenge ROS and promote osteoblast proliferation and osteogenic differentiation through the slow-release effect of POM, providing a feasible strategy for the application of biologically useful bone fillers in bone regeneration. Methods: We synthesized an injectable hydrogel by gelatine methacrylate (GelMA) and POM. The antioxidant capacity and biological properties of the synthesized GelMA/POM hydrogel were tested. Results: In vitro, studies showed that hydrogels can inhibit excessive reactive oxygen species (ROS) and reduce oxidative stress in cells through the beneficial effects of pH-sensitive POM. Osteogenic differentiation assays showed that GelMA/POM had good osteogenic properties with upregulated expression of osteogenic genes (BMP2, RUNX2, Osterix, ALP). Furthermore, RNA-sequencing revealed that activation of the PI3K/Akt signalling pathway in MC3T3-E1 cells with GelMA/POM may be a potential mechanism to promote osteogenesis. In an in vivo study, radiological and histological analyses showed enhanced bone regeneration in diabetic mice, after the application of GelMA/POM. Conclusion: In summary, GelMA/POM hydrogels can enhance bone regeneration by directly scavenging ROS and activating the PI3K/Akt signalling pathway.

[Targeted muscle reinnervation: a surgical technique of human-machine interface for intelligent prosthesis].

Objective: To review targeted muscle reinnervation (TMR) surgery for the construction of intelligent prosthetic human-machine interface, thus providing a new clinical intervention paradigm for the functional reconstruction of residual limbs in amputees. Methods: Extensively consulted relevant literature domestically and abroad and systematically expounded the surgical requirements of intelligent prosthetics, TMR operation plan, target population, prognosis, as well as the development and future of TMR. Results: TMR facilitates intuitive control of intelligent prostheses in amputees by reconstructing the "brain-spinal cord-peripheral nerve-skeletal muscle" neurotransmission pathway and increasing the surface electromyographic signals required for pattern recognition. TMR surgery for different purposes is suitable for different target populations. Conclusion: TMR surgery has been certified abroad as a transformative technology for improving prosthetic manipulation, and is expected to become a new clinical paradigm for 2 million amputees in China.

Deep learning based ultrasonic visualization of distal humeral cartilage for image-guided therapy: a pilot validation study.

Background: Ultrasound is widely used for image-guided therapy (IGT) in many surgical fields, thanks to its various advantages, such as portability, lack of radiation and real-time imaging. This article presents the first attempt to utilize multiple deep learning algorithms in distal humeral cartilage segmentation for dynamic, volumetric ultrasound images employed in minimally invasive surgery. Methods: The dataset, consisting 5,321 ultrasound images were collected from 12 healthy volunteers. These images were randomly split into training and validation sets in an 8:2 ratio. Based on deep learning algorithms, 9 semantic segmentation networks were developed and trained using our dataset at Southern University of Science and Technology Hospital in September 2022. The performance of the networks was evaluated based on their segmenting accuracy and processing efficiency. Furthermore, these networks were implemented in an IGT system to assess their feasibility in 3-dimentional imaging precision. Results: In 2D segmentation, Medical Transformer (MedT) showed the highest accuracy result with a Dice score of 89.4%, however, the efficiency in processing images was relatively lower at 2.6 frames per second (FPS). In 3D imaging, the average root mean square (RMS) between ultrasound (US)-generated models based on the networks and magnetic resonance imaging (MRI)-generated models was no more than 1.12 mm. Conclusions: The findings of this study indicate the technological feasibility of a novel method for real-time visualization of distal humeral cartilage. The increased precision of ultrasound calibration and segmentation are both important approaches to improve the accuracy of 3D imaging.

Cefazolin/BMP-2-Loaded Mesoporous Silica Nanoparticles for the Repair of Open Fractures with Bone Defects.

The study aimed to explore the feasibility of a nanodrug delivery system to treat open fractures with bone defects. We developed a cefazolin (Cef)/bone morphogenetic protein 2 (BMP-2)@mesoporous silica nanoparticle (MSN) delivery system; meanwhile, Cef/MBP-2@ poly(lactic-co-glycolic acid) (PLGA) was also developed as control. For the purpose of determining the osteogenic and anti-inflammatory actions of the nanodelivery system, we cultured bone marrow mesenchymal stem cells (BMSCs) and constructed a bone defect mouse model to evaluate its clinical efficacy. After physicochemical property testing, we determined that MSN had good stability and did not easily accumulate or precipitate and it could effectively prolong the Cef's half-life by nearly eight times. In BMSCs, we found that compared with the PLGA delivery system, MSNs better penetrated into the bone tissue, thus effectively increasing BMSCs' proliferation and migration ability to facilitate bone defect repair. Furthermore, the MSN delivery system could improve BMSCs' mineralization indexes (alkaline phosphatase [ALP], osteocalcin [OCN], and collagen I [Col I]) to effectively improve its osteogenic ability. Moreover, the MSN delivery system could inhibit inflammation in bone defect mice, which was mainly reflected in its ability to reduce the release of IL-1ß and IL-4 and increase IL-10 levels; it could also effectively reduce apoptosis of CD4+ and CD8+ T cells, thus improving their immune function. Furthermore, the percentage of new bones, bone mineral density, trabecular volume, and trabecular numbers in the fracture region were improved in mice treated with MSN, which allowed better repair of bone defects. Hence, Cef/BMP-2@MSN may be feasible for open fractures with bone defects.

Repair of segmental ulnar bone defect in juvenile caused by osteomyelitis with induced membrane combined with tissue-engineered bone: A case report with 4-year follow-up.

INTRODUCTION AND IMPORTANCE: We used induced membrane combined with tissue-engineered bone (TEB) to repair the 14-cm juvenile ulnar defect formed after osteomyelitis debridement. The TEB was completely transformed into autologous bone after 4-year follow-up. CASE PRESENTATION: A 13-year-old male was hospitalized because of right ulna chronic osteomyelitis. After focal debridement, the total length of ular defect was 14 cm. Anti-infective bone cement was filled in the bone defect area. ß-Tricalcium phosphate (ß-TCP) was used as TEB scaffold. Autologous iliac bone marrow stromal cells (BMSCs) were cultured in vitro and were planted on ß-TCP scaffold to form TEB 3 weeks later. 47 months after implantation of TEB, the repaired ulna had continuous and smooth bone cortex, completely ossification of TEB, completely recanalization of medullary cavity. The upper limb function DASH score was 35. CLINICAL DISCUSSION: Masquelet put forward the concept of "induced membrane" and applied this technique on bone defects treatment formed after debridement of osteomyelitis. ß-Tricalcium phosphate (ß-TCP) is artificial bone materials commonly used in clinical. In this case, the seed cells used were autologous BMSCs and the culture medium was autologous serum. Cytokines promoting cell growth and differentiation were not used. CONCLUSION: The results of this case showed that TEB combined with induced membrane could repair ulna segmental bone defects as long as 14 cm in adolescents. This technique gives one alternative method to repair juvenile bone defects caused by osteomyelities of trauma. More clinical cases are needed to verify the effectiveness of this technique in the next.

Distal Humerus Morphological Analysis of Chinese Individuals: A Statistical Shape Modeling Approach.

OBJECTIVE: A detailed analysis of the morphology of distal humeral articulation can help in the creation of anatomic prostheses of hemiarthroplasty. This study used statistical shape modeling to evaluate the 3D morphology of the distal humerus in healthy Chinese individuals and to investigate the proper articular morphology differences. METHODS: A statistical shape model (SSM) of the distal humerus was created using CT scans of 106 survey-confirmed nonpathologic elbows. In addition, the articular components of each principal component (PC) were selected and fitted on the mean mode. The Euclidean point-to-mesh distance of articular modes was calculated as a measurement the proper change in the morphology of the articulation. RESULTS: The first seven PCs jointly accounted for 80.9% of the total variation (44.4%, 12.2%, 7.9%, 5.9%, 4.1%, 3.4% and 3%, respectively). In the mean model, the distance between the medial and lateral epicondyles was 57.4 mm, the width of the articulation was 42.1 mm, and the angle of the transepicondylar line (TEL) and C line was 4.8°. The articular surface differences of the first PC were significant (RMS: 1.43 mm in the -3 SD model and 2.38 mm in the +3 SD model), whereas under other conditions, the differences were not remarkable despite the maximum deformation not exceeding 1 mm. CONCLUSION: A novel method (SSM) was used to evaluate the 3D morphology of the distal humerus in healthy Chinese individuals and investigate the proper articular shape differences. We found the proper shape of articular surface basically transformed into one variation pattern which was relevant to the bone size, even though the morphology of distal humerus possessed complicated variation modes. The findings of this study can be helpful to design the next generation of elbow hemiarthroplasty in the future.

Repair of distal fibular and lateral malleolus defects with individualized 3D-printed titanium alloy prosthesis: The first case report from China.

INTRODUCTION AND IMPORTANCE: This case report describes the reconstruction of the traumatic distal fibular and lateral malleolus defects with a novel method of using individualized 3D printed titanium prosthesis for the first time. CASE PRESENTATION: A 63-year-old male farmer was hospitalized in emergency because of open injury and distal fibular and lateral malleolus defects in the left leg caused by a car accident. 3 months after debridement and latissimus dorsi muscle flap transplantation and skin graft operation, the patient re-hospitalized because of distal fibular and lateral malleolus defect and local pain. We examined the bilateral ankle joint with three-dimensional CT, obtained data about the missing left distal fibular and lateral malleolus through the mirror principle. The corresponding titanium alloy prosthesis then was designed and printed using a 3D metal printer. The patient had no obvious contraindication for surgery, so the prosthesis was surgically implanted. The patient was followed up for 2 years. There was no discomfort at the surgical site. The function of the operated ankle was satisfied by the patient, the AOFAS (American Orthopaedic Foot & Ankle Society) score was 85 (Kitaoka et al., 1994 [1]). CLINICAL DISCUSSION: Individualized 3D printed titanium alloy prosthesis consistent with the anatomical structure of lost distal fibula and lateral malleolus. The proximal end of the prosthesis was designed with four nail holes to install screws to fix the fibula together with it. The lower tibiofibular and talofibular joint surfaces of the prosthesis were designed smoothly. In order to improve the stability of the lower tibiofibular joint, anchors were placed at the attachment of the anterior and posterior tibiofibular ligaments to reconstruct these ligaments. CONCLUSION: The structure and function of the reconstructed distal fibular and the lateral malleous were close to normal. Individualized 3D printed prosthesis might have considerable advantages over traditional treatment methods. The individualized 3D printed titanium alloy prosthesis provides a new method for the repair and reconstruction of similar bone defects. The use of 3D printed prosthesis for surgical repair needs to be further examined in the future through long-term follow-up studies and in more cases.

Schwann cells promote prevascularization and osteogenesis of tissue-engineered bone via bone marrow mesenchymal stem cell-derived endothelial cells.

BACKGROUND: Tissue-engineered bone grafts (TEBGs) that undergo vascularization and neurotization evolve into functioning bone tissue. Previously, we verified that implanting sensory nerve tracts into TEBGs promoted osteogenesis. However, the precise mechanisms and interaction between seed cells were not explored. In this study, we hypothesized that neurotization may influence the osteogenesis of TEBGs through vascularization. METHODS: We cultured rat Schwann cells (SCs), aortic endothelial cells (AECs), and bone marrow-derived mesenchymal stem cells (BM-MSCs) and then obtained BM-MSC-derived induced endothelial cells (IECs) and induced osteoblasts (IOBs). IECs and AECs were cultured in an SC-conditioned medium (SC-CM) to assess proliferation, migration, capillary-like tube formation, and angiogenesis, and the vascular endothelial growth factor (VEGF) levels in the supernatants were detected. We established an indirect coculture model to detect the expression of nestin and VEGF receptors in IECs and tissue inhibitor of metalloproteinase (TIMP)-2 in SCs. Then, SCs, IECs, and IOBs were labeled and loaded into a ß-tricalcium phosphate scaffold to induce prevascularization, and the scaffold was implanted into a 6-mm-long defect of rat femurs. Three groups were set up according to the loaded cells: I, SCs, and IECs (coculture for 3 days) plus IOBs; II, IECs (culture for 3 days) plus IOBs; III, IOBs. Nestin and TIMP-2 expression and osteogenesis of TEBGs were evaluated at 12 weeks post-implantation through histological and radiological assessments. RESULTS: We found that SC-CM promoted IEC proliferation, migration, capillary-like tube formation, and angiogenesis, but no similar effects were observed for AECs. IECs expressed nestin extensively, while AECs barely expressed nestin, and SC-CM promoted the VEGF secretion of IECs. In the coculture model, SCs promoted nestin and VEGF receptor expression in IECs, and IECs inhibited TIMP-2 expression in SCs. The promotion of prevascularized TEBGs by SCs and IECs in group I augmented new bone formation at 6 and 12 weeks. Nestin expression was higher in group I than in the other groups, while TIMP-2 expression was lower at 12 weeks. CONCLUSIONS: This study demonstrated that SCs can promote TEBG osteogenesis via IECs and further revealed the related specific characteristics of IECs, providing preliminary cytological evidence for neurotization of TEBGs.

Biological sealing and integration of a fibrinogen-modified titanium alloy with soft and hard tissues in a rat model.

Percutaneous or transcutaneous devices are important and unique, and the corresponding biological sealing at the skin-implant interface is the key to their long-term success. Herein, we investigated the surface modification to enhance biological sealing, using a metal sheet and screw bonded by biomacromolecule fibrinogen mediated via pre-deposited synthetic macromolecule polydopamine (PDA) as a demonstration. We examined the effects of a Ti-6Al-4V titanium alloy modified with fibrinogen (Ti-Fg), PDA (Ti-PDA) or their combination (Ti-PDA-Fg) on the biological sealing and integration with skin and bone tissues. Human epidermal keratinocytes (HaCaT), human foreskin fibroblasts (HFF) and preosteoblasts (MC3T3-E1), which are closely related to percutaneous implants, exhibited better adhesion and spreading on all the three modified sheets compared with the unmodified alloy. After three-week subcutaneous implantation in Sprague-Dawley (SD) rats, the Ti-PDA-Fg sheets could significantly attenuate the soft tissue response and promote angiogenesis compared with other groups. Furthermore, in the model of percutaneous tibial implantation in SD rats, the Ti-PDA-Fg screws dramatically inhibited epithelial downgrowth and promoted new bone formation. Hence, the covalent immobilization of fibrinogen through the precoating of PDA is promising for enhanced biological sealing and osseointegration of metal implants with soft and hard tissues, which is critical for an orthopedic percutaneous medical device.

3D-bioprinted functional and biomimetic hydrogel scaffolds incorporated with nanosilicates to promote bone healing in rat calvarial defect model.

Three-dimensional (3D) bioprinting is an extremely convenient biofabrication technique for creating biomimetic tissue-engineered bone constructs and has promising applications in regenerative medicine. However, existing bioinks have shown low mechanical strength, poor osteoinductive ability, and lacking a suitable microenvironment for laden cells. Nanosilicate (nSi) has shown to be a promising biomaterial, due to its unique properties such as excellent biocompatibility, degrade into nontoxic products, and with osteoinductive properties, which has been used in bone bioprinting. However, the long term bone healing effects and associating risks, if any, of using nSi in tissue engineering bone scaffolds in vivo are unclear and require a more thorough assessment prior to practical use. Hence, a functional and biomimetic nanocomposite bioink composed of rat bone marrow mesenchymal stem cells (rBMSCs), nSi, gelatin and alginate for the 3D bioprinting of tissue-engineered bone constructs is firstly demonstrated, mimicking the structure of extracellular matrix, to create a conducive microenvironment for encapsulated cells. It is shown that the addition of nSi significantly increases the printability and mechanical strength of fabricated human-scale tissue or organ structures (up to 15 mm height) and induces osteogenic differentiation of the encapsulated rBMSCs in the absence of in vitro osteoinductive factors. A systematic in vivo research of the biomimetic nanocomposite bioink scaffolds is further demonstrated in a rat critical-size (8 mm) bone defect-repair model. The in vivo results demonstrate that the 3D bioprinted nanocomposite scaffolds can significantly promote the bone healing of the rat calvarial defects compared to other scaffolds without nSi or cells, and show rarely side effects on the recipients. Given the above advantageous properties, the 3D bioprinted nanocomposite scaffolds can greatly accelerate the bone healing in critical bone defects, thus providing a clinical potential candidate for orthopedic applications.

Zinc Silicate/Nano-Hydroxyapatite/Collagen Scaffolds Promote Angiogenesis and Bone Regeneration via the p38 MAPK Pathway in Activated Monocytes.

Recent studies show that biomaterials are capable of regulating immune responses to induce a favorable osteogenic microenvironment and promote osteogenesis and angiogenesis. In this study, we investigated the effects of zinc silicate/nanohydroxyapatite/collagen (ZS/HA/Col) scaffolds on bone regeneration and angiogenesis and explored the related mechanism. We demonstrate that 10ZS/HA/Col scaffolds significantly enhanced bone regeneration and angiogenesis in vivo compared with HA/Col scaffolds. ZS/HA/Col scaffolds increased tartrate-resistant acid phosphatase (TRAP)-positive cells, nestin-positive bone marrow stromal cells (BMSCs) and CD31-positive neovessels, and expression of osteogenesis (Bmp-2 and Osterix) and angiogenesis-related (Vegf-α and Cd31) genes increased in nascent bone. ZS/HA/Col scaffolds with 10 wt % ZS activated the p38 signaling pathway in monocytes. The monocytes subsequently differentiated into TRAP+ cells and expressed higher levels of the cytokines SDF-1, TGF-ß1, VEGF-α, and PDGF-BB, which recruited BMSCs and endothelial cells (ECs) to the defect areas. Blocking the p38 pathway in monocytes reduced TRAP+ differentiation and cytokine secretion and resulted in a decrease in BMSC and EC homing and angiogenesis. Overall, these findings demonstrate that 10ZS/HA/Col scaffolds modulate monocytes and, thereby, create a favorable osteogenic microenvironment that promotes BMSC migration and differentiation and vessel formation by activating the p38 signaling pathway.

The effect of fluid shear stress on fibroblasts and stem cells on plane and groove topographies.

In this study, we aimed to study the effect of fluid shear stress on fibroblasts and BMSCs on plane and groove topographies. The results showed that 0.6-Hz stress had the greatest influence on the alignment, polarity, migration and adhesion of fibroblasts on plane by increasing the expression of reoriented actin and vinculin; whereas 1.0-Hz stress promoted differentiation of fibroblasts into myofibroblasts by increasing Col-I and α-SMA expression. Interestingly, under the given frequency stress, the groove structure strengthened the above characteristics of fibroblasts beyond adhesion, and promoted differentiation of BMSCs into myofibroblasts. The above results indicate that 0.6 Hz may improve the implant-tissue sealing, while 1.0-Hz stress probably causes the disordered fiber deposition around implants.

Effects of cyclic fluid stress at different frequencies on behaviors of cells incubated on titanium alloy.

The orthopedic external fixation is always in dynamic mechanical environment with the somatic movement. We used a self-designed mini oscillator to simulate this condition by providing the reciprocating cyclic fluid stress, and observed the behavioral responses of fibroblasts implanted on titanium alloy plane to the stress at different frequencies, including 0.2 Hz, 0.6 Hz, and 1.0 Hz. We found that the cell angle, shape index and expression of vinculin were mostly biphasic-dependent with the increase of frequency, with peaks at 0.6 Hz. Whereas the cell area, expression of Col-I and α-SMA were mainly affected by the 1.0 Hz stress. Interestingly, 1.0 Hz stress also promoted Col-I expression of bone marrow mesenchymal stem cells (BMSCs), although it did not increase α-SMA. These results reveal that 0.6 Hz stress improves the alignment, polarity and adherence of fibroblasts on titanium alloy substrates, thus improving the sealing of implants; the 1.0 Hz force activates the differentiation of fibroblasts into myofibroblasts and increases collagen produced by stem cells, which probably cause the formation of fibrous capsules around implants.

Blockade of adrenergic ß-receptor activation through local delivery of propranolol from a 3D collagen/polyvinyl alcohol/hydroxyapatite scaffold promotes bone repair in vivo.

OBJECTIVES: Activation of the sympathetic system and adrenergic ß-receptors following traumatic bone defects negatively impairs bone regeneration. Whether preventing ß-receptor activation could potentially improve bone defect repair is unknown. In this study, we investigated the effect of systematic administration and local delivery of propranolol through composite scaffolds on bone healing. MATERIALS AND METHODS: Collagen/PVA/propranolol/hydroxyapatite（CPPH）composite scaffolds were fabricated with 3D printing technique and characterized by scanning electron microscope (SEM). Micro-CT analysis and bone formation histology were performed to detect new bone formation. Osteogenic differentiation of bone marrow stromal cells (BMSCs) and osteoclastogenesis of bone marrow monocytes cultured with scaffolds extract were performed for further verification. RESULTS: Intraperitoneal injection of propranolol did not significantly improve bone repair, as indicated by micro-CT analysis and bone formation histology. However, CPPH scaffolds exhibited sustained release of propranolol in vitro and significantly enhanced bone regeneration compared with vehicle collagen/PVA/hydroxyapatite (CPH) scaffolds in vivo. Moreover, in vitro experiments indicated the scaffolds containing propranolol promoted the osteogenic differentiation and migration of rat BMSCs and inhibited osteoclastogenesis by preventing ß-receptor activation. CONCLUSIONS: This study demonstrates that local adrenergic ß-receptor blockade can effectively enhance the treatment of bone defects by stimulating osteogenic differentiation, inhibiting osteoclastogenesis and enhancing BMSCs migration.

Dorsal Root Ganglion Maintains Stemness of Bone Marrow Mesenchymal Stem Cells by Enhancing Autophagy through the AMPK/mTOR Pathway in a Coculture System.

Our previous studies found that sensory nerve tracts implanted in tissue-engineered bone (TEB) could result in better osteogenesis. To explore the mechanism of the sensory nerve promoting osteogenesis in TEB in vitro, a transwell coculture experiment was designed between dorsal root ganglion (DRG) cells and bone marrow mesenchymal stem cells (BMSCs). BMSC proliferation was determined by CCK8 assay, and osteo-, chondro-, and adipogenic differentiation were assessed by alizarin red, alcian blue, and oil red staining. We found that the proliferation and multipotent differentiation of BMSCs were all enhanced in the coculture group compared to the BMSCs group. Crystal violet staining showed that the clone-forming ability of BMSCs in the coculture group was also enhanced and mRNA levels of Sox2, Nanog, and Oct4 were significantly upregulated in the coculture group. Moreover, the autophagy level of BMSCs, regulating their stemness, was promoted in the coculture group, mediated by the AMPK/mTOR pathway. In addition, AMPK inhibitor compound C could significantly downregulate the protein expression of LC3 and the mRNA level of stemness genes in the coculture group. Finally, we found that the NK1 receptor antagonist, aprepitant, could partly block this effect, which indicated that substance P played an important role in the effect. Together, we conclude that DRG could maintain the stemness of BMSCs by enhancing autophagy through the AMPK/mTOR pathway in a transwell coculture system, which may help explain the better osteogenesis after implantation of the sensory nerve into TEB.

Vascularization converts the lineage fate of bone mesenchymal stem cells to endothelial cells in tissue-engineered bone grafts by modulating FGF2-RhoA/ROCK signaling.

The prevascularization of tissue-engineered bone grafts (TEBGs) has been shown to accelerate capillary vessel ingrowth in bone defect remodeling and to enhance new bone formation. However, the exact mechanisms behind this positive effect remain unknown. Here, we report that basic fibroblast growth factor (FGF2)-Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) signaling functions as a molecular switch to regulate the lineage fate of bone mesenchymal stem cells (BMSCs) and that prevascularization promotes the cell fate switch, which contributes to increased bone regeneration with the use of prevascularized TEBGs compared with control TEBGs. Prevascularized TEBGs enhanced the in vivo endothelial differentiation of BMSCs by inhibiting RhoA/ROCK signaling. In vitro data more clearly showed that BMSCs differentiated into von Willebrand factor (vWF)-positive endothelial cells, and FGF2-induced inhibition of RhoA/ROCK signaling played a key role. Our novel findings uncovered a new mechanism that stimulates the increased vascularization of engineered bone and enhanced regeneration by promoting the endothelial differentiation of BMSCs implanted in TEBGs. These results offer a new molecular target to regulate TEBG-induced bone regeneration.

Prevascularization promotes endogenous cell-mediated angiogenesis by upregulating the expression of fibrinogen and connective tissue growth factor in tissue-engineered bone grafts.

BACKGROUND: Vascularization is one of the most important processes in tissue-engineered bone graft (TEBG)-mediated regeneration of large segmental bone defects. We previously showed that prevascularization of TEBGs promoted capillary vessel formation within the defected site and accelerated new bone formation. However, the precise mechanisms and contribution of endogenous cells were not explored. METHODS: We established a large defect (5 mm) model in the femur of EGFP+ transgenic rats and implanted a ß-tricalcium phosphate (ß-TCP) scaffold seeded with exogenous EGFP- cells; the femoral vascular bundle was inserted into the scaffold before implantation in the prevascularized TEBG group. Histopathology and scanning electron microscopy were performed and connective tissue growth factor (CTGF) and fibrin expression, exogenous cell survival, endogenous cell migration and behavior, and collagen type I and III deposition were assessed at 1 and 4 weeks post implantation. RESULTS: We found that the fibrinogen content can be increased at the early stage of vascular bundle transplantation, forming a fibrin reticulate structure and tubular connections between pores of ß-TCP material, which provides a support for cell attachment and migration. Meanwhile, CTGF expression is increased, and more endogenous cells can be recruited and promote collagen synthesis and angiogenesis. By 4 weeks post implantation, the tubular connections transformed into von Willebrand factor-positive capillary-like structures with deposition of type III collagen, and accelerated angiogenesis of endogenous cells. CONCLUSIONS: These findings demonstrate that prevascularization promotes the recruitment of endogenous cells and collagen deposition by upregulating fibrinogen and CTGF, directly resulting in new blood vessel formation. In addition, this molecular mechanism can be used to establish fast-acting angiogenesis materials in future clinical applications.

Novel standardized massive bone defect model in rats employing an internal eight-hole stainless steel plate for bone tissue engineering.

Massive bone defects are a challenge in orthopaedic research. Defective regeneration leads to bone atrophy, non-union of bone, and physical morbidity. Large animals are important models, however, production costs are high, nursing is complex, and evaluation methods are limited. A suitable laboratory animal model is required to explore the underlying molecular mechanism and cellular process of bone tissue engineering. We designed a stainless steel plate with 8 holes; the middle 2 holes were used as a guide to create a standardized critical size defect in the femur of anaesthetized rats. The plate was fixed to the bone using 6 screws, serving as an inner fixed bracket to secure a tricalcium phosphate implant seeded with green fluorescent protein-positive rat bone marrow mesenchymal stem cells within the defect. In some animals, we also grafted a vessel bundle into the lateral side of the implant, to promote vascularized bone tissue engineering. X-ray, microcomputed tomography, and histological analyses demonstrated the stainless steel plate resulted in a stable large segmental defect model in the rat femur. Vascularization significantly increased bone formation and implant degradation. Moreover, survival and expansion of green fluorescent protein-positive seeded cells could be clearly monitored in vivo at 1, 4, and 8 weeks postoperation via fluorescent microscopy. This standardized large segmental defect model in a small animal may help to advance the study of bone tissue engineering. Furthermore, availability of antibodies and genetically modified rats could help to dissect the precise cellular and molecular mechanisms of bone repair.

Nano-biphasic calcium phosphate/polyvinyl alcohol composites with enhanced bioactivity for bone repair via low-temperature three-dimensional printing and loading with platelet-rich fibrin.

BACKGROUND AND AIM: As a newly emerging three-dimensional (3D) printing technology, low-temperature robocasting can be used to fabricate geometrically complex ceramic scaffolds at low temperatures. Here, we aimed to fabricate 3D printed ceramic scaffolds composed of nano-biphasic calcium phosphate (BCP), polyvinyl alcohol (PVA), and platelet-rich fibrin (PRF) at a low temperature without the addition of toxic chemicals. METHODS: Corresponding nonprinted scaffolds were prepared using a freeze-drying method. Compared with the nonprinted scaffolds, the printed scaffolds had specific shapes and well-connected internal structures. RESULTS: The incorporation of PRF enabled both the sustained release of bioactive factors from the scaffolds and improved biocompatibility and biological activity toward bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. Additionally, the printed BCP/PVA/PRF scaffolds promoted significantly better BMSC adhesion, proliferation, and osteogenic differentiation in vitro than the printed BCP/PVA scaffolds. In vivo, the printed BCP/PVA/PRF scaffolds induced a greater extent of appropriate bone formation than the printed BCP/PVA scaffolds and nonprinted scaffolds in a critical-size segmental bone defect model in rabbits. CONCLUSION: These experiments indicate that low-temperature robocasting could potentially be used to fabricate 3D printed BCP/PVA/PRF scaffolds with desired shapes and internal structures and incorporated bioactive factors to enhance the repair of segmental bone defects.