Critical-sized Defect Model Research Articles

Short-Term Evaluation of Cellular Fate in an Ovine Bone Formation Model.

The ovine critical-sized defect model provides a robust preclinical model for testing tissue-engineered constructs for use in the treatment of non-union bone fractures and severe trauma. A critical question in cell-based therapies is understanding the optimal therapeutic cell dose. Key to defining the dose and ensuring successful outcomes is understanding the fate of implanted cells, e.g., viability, bio-distribution and exogenous infiltration post-implantation. This study evaluates such parameters in an ovine critical-sized defect model 2 and 7 days post-implantation. The fate of cell dose and behaviour post-implantation when combined with nanomedicine approaches for multi-model tracking and remote control using external magnetic fields is also addressed. Autologous STRO-4 selected mesenchymal stromal cells (MSCs) were labelled with a fluorescent lipophilic dye (CM-Dil), functionalised magnetic nanoparticles (MNPs) and delivered to the site within a naturally derived bone extracellular matrix (ECM) gel. Encapsulated cells were implanted within a critical-sized defect in an ovine medial femoral condyle and exposed to dynamic gradients of external magnetic fields for 1 h per day. Sheep were sacrificed at 2 and 7 days post-initial surgery where ECM was harvested. STRO-4-positive (STRO-4+) stromal cells expressed osteocalcin and survived within the harvested gels at day 2 and day 7 with a 50% loss at day 2 and a further 45% loss at 7 days. CD45-positive leucocytes were also observed in addition to endogenous stromal cells. No elevation in serum C-reactive protein (CRP) or non-haem iron levels was observed following implantation in groups containing MNPs with or without magnetic field gradients. The current study demonstrates how numbers of therapeutic cells reduce substantially after implantation in the repair site. Cell death is accompanied by enhanced leucocyte invasion, but not by inflammatory blood marker levels. Crucially, a proportion of implanted STRO-4+ stromal cells expressed osteocalcin, which is indicative of osteogenic differentiation. Furthermore, MNP labelling did not alter cell number or result in a further deleterious impact on stromal cells following implantation.

Sustained zinc release in cooperation with CaP scaffold promoted bone regeneration via directing stem cell fate and triggering a pro-healing immune stimuli

Metal ions have been identified as important bone metabolism regulators and widely used in the field of bone tissue engineering, however their exact role during bone regeneration remains unclear. Herein, the aim of study was to comprehensively explore the interactions between osteoinductive and osteo-immunomodulatory properties of these metal ions. In particular, the osteoinductive role of zinc ions (Zn2+), as well as its interactions with local immune microenvironment during bone healing process, was investigated in this study using a sustained Zn2+ delivery system incorporating Zn2+ into β-tricalcium phosphate/poly(L-lactic acid) (TCP/PLLA) scaffolds. The presence of Zn2+ largely enhanced osteogenic differentiation of periosteum-derived progenitor cells (PDPCs), which was coincident with increased transition from M1 to M2 macrophages (Mvarphi s). We further confirmed that induction of M2 polarization by Zn2+ was realized via PI3K/Akt/mTOR pathway, whereas marker molecules on this pathway were strictly regulated by the addition of Zn2+. Synergically, this favorable immunomodulatory effect of Zn2+ further improved the osteogenic differentiation of PDPCs induced by Zn2+ in vitro. Consistently, the spontaneous osteogenesis and pro-healing osteoimmunomodulation of the scaffolds were thoroughly identified in vivo using a rat air pouch model and a calvarial critical-size defect model. Taken together, Zn2+-releasing bioactive ceramics could be ideal scaffolds in bone tissue engineering due to their reciprocal interactions between osteoinductive and immunomodulatory characteristics. Clarification of this synergic role of Zn2+ during osteogenesis could pave the way to develop more sophisticated metal-ion based orthopedic therapeutic strategies.

Gellan gum/alginate-based Ca-enriched acellular bilayer hydrogel with robust interface bonding for effective osteochondral repair

The treatment of osteochondral (OC) defects remains challenging because of the lack of economical and feasible therapeutic strategies for OC repair and reconstruction. In this study, we report an integrated bilayer hydrogel with robust interface binding force (40 kPa) by facilitating the diffusion of calcium ions to the secondary crosslink of the bilayer hydrogel, in which gellan gum and sodium alginate acted as the chondral layer, gellan gum and hydroxyapatite acted as subchondral layer. This integrated construct has high cytocompatibility, and can seed with mesenchymal stem cells (MSCs) related to different functional protein expression for cartilage and bone formation, respectively. Furthermore, in the rabbit critical-sized osteochondral defect model (4.0 mm in diameter and 8.0 mm in depth), the calcium enriched hydrogel act as a calcium reservoir, promote neovascularization at week 4, and repair the critical defect at week 8, demonstrating the feasible preparation of an acellular hydrogel for OC repair.

PTH1-34 improves devitalized allogenic bone graft healing in a murine femoral critical size defect

The treatment of large segmental defects of long bones resulting from trauma, infection, or bone tumor resections is a major challenge for orthopedic surgeons. The reconstruction of bone defects with acellular allografts can be used as an osteoconductive approach. However, devitalized allografts are associated with high rates of clinical failure as a result of poor intrinsic osteoinduction properties and a lack of further remodeling. Nevertheless, evidence suggests that due to its anabolic properties, teriparatide (PTH1-34) could be effective as an adjuvant therapy for massive allograft healing. Therefore, our goal was to investigate in a murine critical-sized defect model whether the intermittent administration of PTH1-34 improves the incorporation and revitalization of acellular structural bone allografts. Thus, a 2.5-mm critical-sized defect was established in the right femur of C57BL/6 mice, followed by the reconstruction with a devitalized cortical structural allograft. A titanium micro locking plate was applied to the anterior femoral surface and secured in place with self-tapping locking screws. Subsequently, daily doses of PTH1–34 (30, and 40 µg/kg) or saline were administered to the mice for 14 days after surgery. The mice were maintained without PTH1–34 therapy for an additional 7 days before being euthanized at 3 weeks post-surgery. Bone graft consolidation was assessed on radiographic images and by histomorphometric analysis. Additionally, to determine the frequency of osteoprogenitor cells in the bone marrow and their in vitro osteogenic capacity, stromal cells were isolated from the bone marrow of animals treated with 30 or 40 µg/kg/day of PTH1-34 following the same protocol used for the experimental animals. Our results suggest that intermittent PTH1-34 treatment at 30 µg/kg/day after femoral allograft reconstruction surgery accelerated the healing process as evidenced by new bone formation induced on endosteal and periosteal surfaces, enhanced revitalization of allogeneic graft, and increased frequency and osteogenic capacity of bone marrow stromal cells (BMSC). These findings should encourage further studies aimed at investigating the potential therapeutic use of intermittent PTH1-34, specifically with regards to the optimal dosing regimen in clinically challenging orthopedic scenarios.

Regional gene therapy for bone healing using a 3D printed scaffold in a rat femoral defect model.

At the present time there are no consistently satisfactory treatment options for some challenging bone loss scenarios. We have previously reported on the properties of a novel 3D-printed hydroxyapatite-composite material in a pilot study, which demonstrated osteoconductive properties but was not tested in a rigorous, clinically relevant model. We therefore utilized a rat critical-sized femoral defect model with a scaffold designed to match the dimensions of the bone defect. The scaffolds were implanted in the bone defect after being loaded with cultured rat bone marrow cells (rBMC) transduced with a lentiviral vector carrying the cDNA for BMP-2. This experimental group was compared against 3 negative and positive control groups. The experimental group and positive control group loaded with rhBMP-2 demonstrated statistically equivalent radiographic and histologic healing of the defect site (p > 0.9), and significantly superior to all three negative control groups (p < 0.01). However, the healed defects remained biomechanically inferior to the unoperated, contralateral femurs (p < 0.01). When combined with osteoinductive signals, the scaffolds facilitate new bone formation in the defect. However, the scaffold alone was not sufficient to promote adequate healing, suggesting that it is not substantially osteoinductive as currently structured. The combination of gene therapy with 3D-printed scaffolds is quite promising, but additional work is required to optimize scaffold geometry, cell dosage and delivery.

3D-printed scaffold combined to 2D osteoinductive coatings to repair a critical-size mandibular bone defect.

The reconstruction of large bone defects (12 cm3) remains a challenge for clinicians. We developed a new critical-size mandibular bone defect model on a minipig, close to human clinical issues. We analyzed the bone reconstruction obtained by a 3D-printed scaffold made of clinical-grade polylactic acid (PLA), coated with a polyelectrolyte film delivering an osteogenic bioactive molecule (BMP-2). We compared the results (computed tomography scans, microcomputed tomography scans, histology) to the gold standard solution, bone autograft. We demonstrated that the dose of BMP-2 delivered from the scaffold significantly influenced the amount of regenerated bone and the repair kinetics, with a clear BMP-2 dose-dependence. Bone was homogeneously formed inside the scaffold without ectopic bone formation. The bone repair was as good as for the bone autograft. The BMP-2 doses applied in our study were reduced 20- to 75-fold compared to the commercial collagen sponges used in the current clinical applications, without any adverse effects. Three-dimensional printed PLA scaffolds loaded with reduced doses of BMP-2 may be a safe and simple solution for large bone defects faced in the clinic.

In Vivo Evaluation of the Regenerative Capability of Glycylglycine Ethyl Ester-Substituted Polyphosphazene and Poly(lactic-co-glycolic acid) Blends: A Rabbit Critical-Sized Bone Defect Model.

In an effort to understand the biological capability of polyphosphazene-based polymers, three-dimensional biomimetic bone scaffolds were fabricated using the blends of poly[(glycine ethylglycinato)75(phenylphenoxy)25]phosphazene (PNGEGPhPh) and poly(lactic-co-glycolic acid) (PLGA), and an in vivo evaluation was performed in a rabbit critical-sized bone defect model. The matrices constructed from PNGEGPhPh-PLGA blends were surgically implanted into 15 mm critical-sized radial defects of the rabbits as structural templates for bone tissue regeneration. PLGA, which is the most commonly used synthetic bone graft substitute, was used as a control in this study. Radiological and histological analyses demonstrated that PNGEGPhPh-PLGA blends exhibited favorable in vivo biocompatibility and osteoconductivity, as the newly designed matrices allowed new bone formation to occur without adverse immunoreactions. The X-ray images of the blends showed higher levels of radiodensity than that of the pristine PLGA, indicating higher rates of new bone formation and regeneration. Micro-computed tomography quantification revealed that new bone volume fractions were significantly higher for the PNGEGPhPh-PLGA blends than for the PLGA controls after 4 weeks. The new bone volume increased linearly with increasing time points, with the new tissues observed throughout the defect area for the blend and only at the implant site's extremes for the PLGA control. Histologically, the polyphosphazene system appeared to show tissue responses and bone ingrowths superior to PLGA. By the end of the study, the defects with PNGEGPhPh-PLGA scaffolds exhibited evidence of effective bone tissue ingrowth and minimal inflammatory responses. Thus, polyphosphazene-containing biomaterials have excellent translational potential for use in bone regenerative engineering applications.

Magneto-sensitive decellularized bone matrix with or without low frequency-pulsed electromagnetic field exposure for the healing of a critical-size bone defect

Bioactive ECM-based materials mimic the complex composition and structure of natural tissues. Decellularized cancellous bone matrix (DBM) has potential for guiding new bone formation and accelerating the regeneration process. On the other hand, low frequency-pulsed electromagnetic field (LF-PEMF) has been shown to enhance the regeneration capacity of bone defects. The present study sought to explore the feasibility of using DBM and DBM/MNP, and LF-PEMF for treating critical-size bone defects. Firstly, decellularization protocol was optimized to obtain a bioactive DBM, then MNPs were incorporated. Later, the physical, chemical and biological properties of DBM and DBM/MNP were assessed in vitro. MNPs homogeneously distributed into the DBM were not found to be toxic to human osteoblast cultures. Finally, an in vivo study was carried out with DBM and DBM/MNP composites in a bilateral critical-size rat cranial defect model (n = 48) with or without LF-PEMF exposure for 45 and 90 days. The histomorphometric and radiographic evaluations revealed that, while the collagen (positive control) and Sham (negative control) groups showed high incidence of fibrous connective tissue together with low level of osteogenic activity, both the DBM and DBM/MNP-grafted groups significantly promoted new bone tissue formation and angiogenesis, by the appropriate use of LF-PEMF for 90 days.

S53P4 bioactive glass scaffolds induce BMP expression and integrative bone formation in a critical-sized diaphysis defect treated with a single-staged induced membrane technique

Critical-sized diaphysis defects are complicated by inherent sub-optimal healing conditions. The two-staged induced membrane technique has been used to treat these challenging defects since the 1980’s. It involves temporary implantation of a membrane-inducing spacer and subsequent bone graft defect filling. A single-staged, graft-independent technique would reduce both socio-economic costs and patient morbidity. Our aim was to enable such single-staged approach through development of a strong bioactive glass scaffold that could replace both the spacer and the graft filling. We constructed amorphous porous scaffolds of the clinically used bioactive glass S53P4 and evaluated them in vivo using a critical-sized defect model in the weight-bearing femur diaphysis of New Zealand White rabbits. S53P4 scaffolds and standard polymethylmethacrylate spacers were implanted for 2, 4, and 8 weeks. Induced membranes were confirmed histologically, and their osteostimulative activity was evaluated through RT-qPCR of bone morphogenic protein 2, 4, and 7 (BMPs). Bone formation and osseointegration were examined using histology, scanning electron microscopy, energy-dispersive X-ray analysis, and micro-computed tomography imaging. Scaffold integration, defect union and osteosynthesis were assessed manually and with X-ray projections. We demonstrated that S53P4 scaffolds induce osteostimulative membranes and produce osseointegrative new bone formation throughout the scaffolds. We also demonstrated successful stable scaffold integration with early defect union at 8 weeks postoperative in critical-sized segmental diaphyseal defects with implanted sintered amorphous S53P4 scaffolds. This study presents important considerations for future research and the potential of the S53P4 bioactive glass as a bone substitute in large diaphyseal defects. Statement of significanceSurgical management of critical-sized diaphyseal defects involves multiple challenges, and up to 10% result in delayed or non-union. The two-staged induced membrane technique is successfully used to treat these defects, but it is limited by the need of several procedures and bone graft. Repeated procedures increase costs and morbidity, while grafts are subject to donor-site complications and scarce availability. To transform this two-staged technique into one graft-independent procedure, we developed amorphous porous scaffolds sintered from the clinically used bioactive glass S53P4. This work constitutes the first evaluation of such scaffolds in vivo in a critical-sized diaphyseal defect in the weight-bearing rabbit femur. We provide important knowledge and prospects for future development of sintered S53P4 scaffolds as a bone substitute.

Hypoxia-mimicking 3D bioglass-nanoclay scaffolds promote endogenous bone regeneration

Large bone defect repair requires biomaterials that promote angiogenesis and osteogenesis. In present work, a nanoclay (Laponite, XLS)-functionalized 3D bioglass (BG) scaffold with hypoxia mimicking property was prepared by foam replication coupled with UV photopolymerization methods. Our data revealed that the incorporation of XLS can significantly promote the mechanical property of the scaffold and the osteogenic differentiation of human adipose mesenchymal stem cells (ADSCs) compared to the properties of the neat BG scaffold. Desferoxamine, a hypoxia mimicking agent, encourages bone regeneration via activating hypoxia-inducible factor-1 alpha (HIF-1α)-mediated angiogenesis. GelMA-DFO immobilization onto BG-XLS scaffold achieved sustained DFO release and inhibited DFO degradation. Furthermore, in vitro data demonstrated increased HIF-1α and vascular endothelial growth factor (VEGF) expressions on human adipose mesenchymal stem cells (ADSCs). Moreover, BG-XLS/GelMA-DFO scaffolds also significantly promoted the osteogenic differentiation of ADSCs. Most importantly, our in vivo data indicated BG-XLS/GelMA-DFO scaffolds strongly increased bone healing in a critical-sized mouse cranial bone defect model. Therefore, we developed a novel BG-XLS/GelMA-DFO scaffold which can not only induce the expression of VEGF, but also promote osteogenic differentiation of ADSCs to promote endogenous bone regeneration.

Shapable bulk agarose–gelatine–hydroxyapatite–minocycline nanocomposite fabricated using a mineralizing system aided with electrophoresis for bone tissue regeneration

To develop a shapable bulk antibacterial nanocomposite biomaterial for bone regeneration. A bulk agarose–gelatine hydrogel was mineralized using a hydrogel mineralizing system aided with electrophoresis, and the mineralized hydrogel was loaded with minocycline to obtain the agarose–gelatine–hydroxyapatite–minocycline nanocomposite. The nanocomposite had a large Brunauer–Emmett–Teller surface area of 44.4518 m2 g−1 and a high porosity of 76.9%. Hydroxyapatite crystals were well developed in the hydrogel matrix and exhibited a hybrid structure of microscale and nanoscale motifs. The addition of minocycline resulted in a continuous antibiotic release, inhibiting the growth of Staphylococcus aureus over 2 weeks in vitro. Exposed to rabbit bone marrow mesenchymal stem cells, the nanocomposite revealed good cytocompatibility in vitro. Furthermore, the biomaterial could effectively enhance the bone regeneration in a critical-size rabbit cranial defect model in vivo. These findings depicted that the nanocomposite, with good biocompatibility and good antibacterial property, is a promising candidate for future clinical application in bone tissue engineering or as a prospective bone replacement biomaterial.

A partially demineralized allogeneic bone graft: in vitro osteogenic potential and preclinical evaluation in two different intramembranous bone healing models

In skeletal surgical procedures, bone regeneration in irregular and hard-to-reach areas may present clinical challenges. In order to overcome the limitations of traditional autologous bone grafts and bone substitutes, an extrudable and easy-to-handle innovative partially demineralized allogenic bone graft in the form of a paste has been developed. In this study, the regenerative potential of this paste was assessed and compared to its clinically used precursor form allogenic bone particles. Compared to the particular bone graft, the bone paste allowed better attachment of human mesenchymal stromal cells and their commitment towards the osteoblastic lineage, and it induced a pro-regenerative phenotype of human monocytes/macrophages. The bone paste also supported bone healing in vivo in a guide bone regeneration model and, more interestingly, exhibited a substantial bone-forming ability when implanted in a critical-size defect model in rat calvaria. Thus, these findings indicate that this novel partially demineralized allogeneic bone paste that combines substantial bone healing properties and rapid and ease-of-use may be a promising alternative to allogeneic bone grafts for bone regeneration in several clinical contexts of oral and maxillofacial bone grafting.

The effects of high fat diet, bone healing, and BMP-2 treatment on endothelial cell growth and function.

Angiogenesis is a vital process during the regeneration of bone tissue. The aim of this study was to investigate angiogenesis at the fracture site as well as at distal locations from obesity-induced type 2 diabetic mice that were treated with bone morphogenetic protein-2 (BMP-2, local administration at the time of surgery) to heal a femoral critical sized defect (CSD) or saline as a control. Mice were fed a high fat diet (HFD) to induce a type 2 diabetic-like phenotype while low fat diet (LFD) animals served as controls. Endothelial cells (ECs) were isolated from the lungs (LECs) and bone marrow (BMECs) 3weeks post-surgery, and the fractured femurs were also examined. Our studies demonstrate that local administration of BMP-2 at the fracture site in a CSD model results in complete bone healing within 3weeks for all HFD mice and 66.7% of LFD mice, whereas those treated with saline remain unhealed. At the fracture site, vessel parameters and adipocyte numbers were significantly increased in BMP-2 treated femurs, irrespective of diet. At distal sites, LEC and BMEC proliferation was not altered by diet or BMP-2 treatment. HFD increased the tube formation ability of both LECs and BMECs. Interestingly, BMP-2 treatment at the time of surgery reduced tube formation in LECs and humeri BMECs. However, migration of BMECs from HFD mice treated with BMP-2 was increased compared to BMECs from HFD mice treated with saline. BMP-2 treatment significantly increased the expression of CD31, FLT-1, and ANGPT2 in LECs and BMECs in LFD mice, but reduced the expression of these same genes in HFD mice. To date, this is the first study that depicts the systemic influence of fracture surgery and local BMP-2 treatment on the proliferation and angiogenic potential of ECs derived from the bone marrow and lungs.

Application of bioluminescence resonance energy transfer-based cell tracking approach in bone tissue engineering

Bioluminescent imaging (BLI) has emerged as a popular in vivo tracking modality in bone regeneration studies stemming from its clear advantages: non-invasive, real-time, and inexpensive. We recently adopted bioluminescence resonance energy transfer (BRET) principle to improve BLI cell tracking and generated the brightest bioluminescent signal known to date, which thus enables more sensitive real-time cell tracking at deep tissue level. In the present study, we brought BRET-based cell tracking strategy into the field of bone tissue engineering for the first time. We labeled rat mesenchymal stem cells (rMSCs) with our in-house BRET-based GpNLuc reporter and evaluated the cell tracking efficacy both in vitro and in vivo. In scaffold-free spheroid 3D culture system, using BRET-based GpNLuc labeling resulted in significantly better correlation to cell numbers than a fluorescence based approach. In scaffold-based 3D culture system, GpNLuc-rMSCs displayed robust bioluminescence signals with minimal background noise. Furthermore, a tight correlation between BLI signal and cell number highlighted the robust reliability of using BRET-based BLI. In calvarial critical sized defect model, robust signal and the consistency in cell survival evaluation collectively supported BRET-based GpNLuc labeling as a reliable approach for non-invasively tracking MSC. In summary, BRET-based GpNLuc labeling is a robust, reliable, and inexpensive real-time cell tracking method, which offers a promising direction for the technological innovation of BLI and even non-invasive tracking systems, in the field of bone tissue engineering.

Digital light processing strength-strong ultra-thin bioceramic scaffolds for challengeable orbital bone regeneration and repair in Situ

The orbital fracture regeneration and reconstruction is still a catastrophic challenge due to the extremely thin anatomical structure and different defect shapes in various clinical conditions. The conventional biomaterials and preparation technique are often inefficient in satisfying porous scaffold architecture designs with respect to their required structural stability and biological performances. Here we developed the ultrathin scaffolds with high- precision pore structure and appreciable osteogenic activity for orbital bone repair via digital light processing (DLP) stereolithography. The dilute magnesium-substituting wollastonite (CSi-Mg) bioceramic scaffolds of ~1.5 mm in thickness and over 60% in porosity were investigated systematically, and compared with another calcium-magnesium silicate (i.e. akermanite) and the pure wollastonite porous counterparts. The results showed that the flexural strengths (~6−22 MPa) and in vitro biodegradation of such bioceramic scaffolds could be tuned during their chemical composition (i.e. Mg content) design stage, and the CSi-Mg scaffolds were also beneficial for osteogenic cell activity in vitro. In particular, the CSi-Mg scaffolds could readily enhance the new bone ingrowth at 4 weeks of implantation in a critical-sized rabbit orbital bone defect model, and such strength-strong scaffolds exhibited outstanding orbital defect repair efficacy after 12 weeks of implantation, in comparison with the structural instability of other scaffolds. Basically, it is demonstrated that the dilute magnesium doping in wollastonite bioceramic can be favorable for developing the mechanically reliable scaffolds with high porosity and bioactivity but quite thin wall morphology, and meanwhile the DLP-assisted additive manufacturing help us to optimize such porous bioceramic design with respect to their required structural stability for orbital bone reconstruction.

A multi-functionalized calcitriol sustainable delivery system for promoting osteoporotic bone regeneration both in vitro and in vivo

Among metabolic and systemic bone diseases, osteoporosis is known as one of the most progressive disease characterized by bone microstructural deterioration and bone mass reduction. Calcitriol (Cal) is widely accepted as a fat-soluble small molecule drug with reported anti-osteoporotic activity. However, delivery systems capable of local sustained release of small molecule drugs remain a significant challenge. Herein, based on poly(D, L-lactide)-poly(ethylene glycol)-poly(D, L-lactide) (PDLLA-PEG-PDLLA) hydrogels, we developed a thermosensitive and multifunctional sustainable delivery system by doubly integrating with polydopamine modified nano-hydroxyapatite (HA-D) and calcitriol-loaded amino-terminated poly(ethylene glycol)-block-poly(epsilon-caprolactone) (PCL-PEG-NH2) micelles to realize the long-term delivery of small molecule drugs. The sustained release of Cal from the system was associate with triple controlled release effects including the protective screen of the hydrogels over micelles, the reservoir effect of the HA nanoparticles for Cal, and the encapsulation of Cal in micelles. In vitro, results from the present study demonstrated that sustainable delivery system effectively activated osteogenesis, proliferation and migration in these bone mesenchymal stromal cells of ovariectomized rats (OVX-rBMSCs). In vivo, the composite Cal-loaded system was verified to promote bone regeneration in the critical-size femoral epicondyle bone defect model. Meanwhile, little cytotoxicity and appropriate degradation rate of the composite Cal delivery system were revealed. Thus, incorporating Cal into a multifunctional delivery system to perform a long-acting drug delivery in an injectable hydrogel form is a promising therapeutic to improve anti-osteoporotic ability and promote bone regeneration of osteoporosis.

3D-printed bioactive and biodegradable hydrogel scaffolds of alginate/gelatin/cellulose nanocrystals for tissue engineering

The 3D-printed hybrid biodegradable hydrogels composed of alginate, gelatin, and cellulose nanocrystals (CNCs) were prepared to provide a favorable environment for cell proliferation, adhesion, nutrients exchange, and matrix mineralization for bone tissue engineering (BTE) applications. The hybrid scaffolds exhibited enhanced mechanical strength compared to the pure polymer scaffolds. The biocompatibility, differentiation potential, and bone regeneration potential of the printed scaffolds were evaluated by DAPI staining, live-dead assay, alizarin Red-S (ARS) staining, real-time PCR (qRT-PCR), and μCT analysis, respectively. Enhanced cell proliferation has occurred 1% CNC/Alg/Gel scaffolds compared to the control. The cells were adequately adhered to the scaffold and exhibited the flattened structure. Improved mineralization was observed in the 1% CNC/Alg/Gel scaffolds' presence than the control, showing their mineralization efficiency. A significant enhancement in the expression of osteogenic-specific gene markers (Runx2, ALP, BMP-2, OCN, OPN, BSP, and COL1) has occurred with 1% CNC/Alg/Gel than the control, indicating their osteogenic potential. Furthermore, enhanced bone formation was observed in the scaffolds treated groups than the control in the calvaria critical-sized defects (CCD-1) model, suggesting their improved bone regeneration potential. Therefore, the fabricated scaffolds have the potential to explore as a biomaterial for tissue engineering.

Baghdadite Ceramics Prevent Senescence in Human Osteoblasts and Promote Bone Regeneration in Aged Rats.

Bone fractures and critical-sized bone defects present significant health threats for the elderly who have limited capacity for regeneration due to the presence of functionally compromised senescent cells. A wide range of synthetic materials has been developed to promote the regeneration of critical-sized bone defects, but it is largely unknown if a synthetic biomaterial (scaffold) can modulate cellular senescence and improve bone regeneration in aged scenarios. The current study investigates the interaction of Baghdadite (Ca3ZrSi2O9) ceramic scaffolds with senescent human primary osteoblast-like cells (HOBs) and its bone regeneration capacity in aged rats. A senescent HOB model was established by repeatedly passaging HOBs till passage 7 (P7). Compared to the clinically used hydroxyapatite/tricalcium phosphate (HA/TCP), Baghdadite prevented senescence induction in P7 HOBs and markedly negated the paracrine effect of P7 HOB secretomes that mediated the up-regulations of cellular senescence-associated gene expression levels in P2 HOBs. We further demonstrated that conditioned media extracted from Baghdadite corrected the dysfunctional mitochondria in P7 HOBs. In vivo, the bone regeneration capacity was enhanced when 3D printed Baghdadite scaffolds were implanted in a calvaria critical-sized bone defect model in both young and aged rats compared to HA/TCP scaffolds, but a better effect was observed in aged rats than in young rats. This study suggests that Baghdadite ceramic represents a novel and promising biomaterial approach to promote bone regeneration capacity in the elderly by providing an anti-senescent microenvironment.

Application of a New Type of Natural Calcined Bone Repair Material Combined with Concentrated Growth Factors in Bone Regeneration in Rabbit Critical-Sized Calvarial Defect.

Purpose This study is aimed at investigating bone regeneration in critical-sized defects in rabbit calvarium using a novel nano- (n-) hydroxyapatite hybrid scaffold with concentrated growth factors (CGFs). Methods Twenty-four male adult rabbits were chosen to establish a critical-sized bone defect model and randomly divided into two groups. Two defects of 15 mm diameter each were created in the parietal bone of each animal. Group A had n-hydroxyapatite hybrid scaffold placed in the experimental defect on the right, and the left defect was unfilled as blank. Group B had hydroxyapatite hybrid scaffold mixed with CGF placed in the right defect and CGF on the left. Six animals in each group were sacrificed after 6 and 12 weeks. Cone-beam computed tomography system scanning and hematoxylin and eosin (HE) staining were used to detect osteogenesis within the defects. Results The treatment with n-hydroxyapatite hybrid scaffold along with CGF resulted in a significantly higher amount of new bone at 6 and 12 weeks compared to the treatment with CGF alone and the controls. No apparent inflammation and foreign body reaction were observed through HE staining. Conclusions The new synthesized n-hydroxyapatite hybrid scaffold and CGF can be applied for bone defect regeneration to promote the process to a certain extent.

Dentin-Derived Inorganic Minerals Promote the Osteogenesis of Bone Marrow-Derived Mesenchymal Stem Cells: Potential Applications for Bone Regeneration.

Background Oral and maxillofacial bone loss is highly prevalent among populations, and nowadays, increased attention has been focused on dentin derivatives serving as desirable graft materials for bone regeneration. In this study, dentin-derived inorganic mineral (DIM) was fabricated with a high-temperature calcination technique and the effects of DIM on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) and the bone formation were elucidated. Methods The effects of DIM on BMMSC proliferation and apoptosis capacity were evaluated by CCK-8, flow cytometry, and EdU assays. Alkaline phosphatase (ALP) activity detection, ALP staining, alizarin red staining, and osteogenic marker expression analysis were performed to investigate the influence of DIM on the osteogenic differentiation of BMMSCs, as well as the relevant signal mechanisms. The model of critical-sized defects in the calvarium of rats was constructed for exploring the in vivo efficiency of DIM on bone regeneration. Results Cell viability assays indicated that DIM had no cytotoxicity. BMMSCs cultured with DIM presented a higher level of osteogenic differentiation ability than those in the control group. The activation in ERK and p38 signals was detected in DIM-treated BMMSCs, and both pathways and osteogenic process were suppressed while using ERK inhibitor U0126 and p38 inhibitor SB203580, respectively. Furthermore, the animal experiments revealed that DIM could dramatically enhance new bone formation compared to the control group. Conclusion DIM could promote BMMSC osteogenic differentiation via triggering the ERK and p38 MAPK signaling pathways and might be a novel predictable material for facilitating bone formation.