Critical-sized Calvaria Defects Research Articles

Enhancing calvarial defects repair with PDGF-BB mimetic peptide hydrogels

Addressing bone defects represents a significant challenge to public health. Localized delivery of growth factor has emerged as promising approach for bone regeneration. However, the clinical application of Platelet-Derived Growth Factor (PDGF) is hindered by its high cost and short half-life. In this work, we introduce the application of PDGF-mimicking peptide (PMP1) hydrogels for calvarial defect restoration, showcasing their remarkable effectiveness. Through osteogenic differentiation assays and q-PCR analyses, we demonstrate PMP1's substantial capacity to enhance osteogenic differentiation of bone marrow mesenchymal stem cell (BMSC), leading to increased expression of crucial osteogenic genes. Further molecular mechanistic investigations reveal PMP1's activation of the PI3K-AKT-mTOR signaling pathway, a key element of its osteogenic effect. In vivo experiments utilizing a rat calvaria critical-sized defect model underscore the hydrogels' exceptional ability to accelerate new bone formation, thereby significantly advancing the restoration of calvaria defects. This research provides a promising bioactive material for bone tissue regeneration.

Comparison of different bone substitutes in the repair of rat calvaria critical size defects: questioning the need for alveolar ridge presentation.

This study aimed to evaluate the effectiveness of biomaterials in bone healing of critical bone defects created by piezoelectric surgery in rat calvaria. Histomorphologic analysis was performed to assess bone regeneration and tissue response. Fifty animals were randomized into five groups with one of the following treatments: Control group (n = 10), spontaneous blood clot formation with no bone fill; BO group (Bio-Oss, Geistlich Pharma; n = 10), defects were filled with bovine medullary bone substitute; BF group (Bonefill, Bionnovation; n = 10), defects were filled with bovine cortical bone substitute; hydroxyapatite group (n = 10), defects were filled with hydroxyapatite; calcium sulfate group (n = 10), defects were filled with calcium sulfate. Five animals from each group were euthanized at 30 and 45 days. The histomorphometry calculated the percentage of the new bone formation in the bone defect. All data obtained were evaluated statistically considering P < .05 as statistically significant. The results demonstrated the potential of all biomaterials for enhancing bone regeneration. The findings showed no statistical differences between all the biomaterials at 30 and 45 days including the control group without bone grafting. In conclusion, the tested biomaterials presented an estimated capacity of osteoconduction, statistically nonsignificant between them. In addition, the selection of biomaterial should consider the specific clinical aspect, resorption rates, size of the particle, and desired bone healing responses. It is important to emphasize that in some cases, using no bone filler might provide comparable results with reduced cost and possible complications questioning the very frequent use of ridge presentation procedures.

In Vivo Osteogenic and Angiogenic Properties of a 3D-Printed Isosorbide-Based Gyroid Scaffold Manufactured via Digital Light Processing.

Osteogenic and angiogenic properties of synthetic bone grafts play a crucial role in the restoration of bone defects. Angiogenesis is recognised for its support in bone regeneration, particularly in larger defects. The objective of this study is to evaluate the new bone formation and neovascularisation of a 3D-printed isosorbide-based novel CSMA-2 polymer in biomimetic gyroid structures. The gyroid scaffolds were fabricated by 3D printing CSMA-2 polymers with different hydroxyapatite (HA) filler concentrations using the digital light processing (DLP) method. A small animal subcutaneous model and a rat calvaria critical-size defect model were performed to analyse tissue compatibility, angiogenesis, and new bone formation. The in vivo results showed good biocompatibility of the 3D-printed gyroid scaffolds with no visible prolonged inflammatory reaction. Blood vessels were found to infiltrate the pores from day 7 of the implantation. New bone formation was confirmed with positive MT staining and BMP-2 expression, particularly on scaffolds with 10% HA. Bone volume was significantly higher in the CSMA-2 10HA group compared to the sham control group. The results of the subcutaneous model demonstrated a favourable tissue response, including angiogenesis and fibrous tissue, indicative of the early wound healing process. The results from the critical-size defect model showcased new bone formation, as confirmed by micro-CT imaging and immunohistochemistry. The combination of CSMA-2 as the 3D printing material and the gyroid as the 3D structure was found to support essential events in bone healing, specifically angiogenesis and osteogenesis.

Evaluation of the osteogenic potential of demineralized and decellularized bovine bone granules following implantation in rat calvaria critical-size defect model.

The aim of this study was to compare the ability of demineralized (DMB) and decellularized (DCC) bovine bone granules to support bone regeneration in rat calvaria critical-size defects. DMB and DCC were prepared using a previously published method. The granule size used ranged between 500 and 750 μm. A total of forty-eight Sprague-Dawley rats were divided into two groups (n = 24). A pair of 5 mm diameter defects were created on the calvaria of the rats in the right and left parietal bone in both groups. Group A animals received DMB granules and Group B received DCC granules in the right parietal defect side while the left parietal untreated defect acted as sham surgery for both groups. Four animals per group were euthanized in a CO2 chamber at day 7, 14 and 21 post-surgery and the calvaria implantation site biopsy harvested was subjected to osteogenic gene expression analysis. Another four animals per group were euthanized at days 15, 30 and 60 post surgery and the calvaria implantation site biopsy harvested was subjected to histological, immunohistochemistry, RAMAN spectroscopy and Micro-CT analysis at the mentioned time points. Statistical analysis was conducted using t-tests and ANOVA. Histomorphometry showed significantly higher new bone formation in the DCC sites (p<0.05) compared to DMB. Both DMB and DCC implantation sites showed distinct staining for osteocalcin and osteopontin proteins compared to their respective sham sites. By day 21 after implantation, DCC sites demonstrated significantly elevated mRNA levels of osteonectin (p<0.001), osteopontin (p<0.001), osteocalcin (p<0.0001), ALP (p<0.01), and BMP-2 (p<0.001) compared to DMB. However, VEGF expression showed no significant differences at this time point between the two groups. Micro-CT analysis also showed enhanced defect closure and higher bone density in DCC implanted sites while RAMAN spectra demonstrated increased abundance of collagen and bone minerals, especially, PO43- ions than DMB. In conclusion, both DMB and DCC granules demonstrated favorable osteogenic potential in critical-sized defects, with DCC exhibited superior osteoconductive, osteoinductive and osteogenesis properties.

Chondrocytes supplemented to bone graft-containing scaffolds expedite cranial defect repair

Critical maxillofacial bone fractures do not heal spontaneously, thus, often there is a need to facilitate repair via surgical intervention. Gold standard approaches, include the use of autologous bone graft, or devices supplemented with osteogenic growth factors and bone substitutes. This research aimed to employ a critical size calvaria defect model, to determine if the addition of chondrocytes to collagen-containing bone graft substitute, may expedite bone repair. As such, using a critical size rat calvaria defect, we implanted a collagen scaffold containing bone graft substitute (i.e., Bone graft scaffold, BG) or BG supplemented with costal chondrocytes (cBG). The rats were subjected to live CT imaging at 1, 6, 9, and 12 weeks following the surgical procedure and sacrificed for microCT imaging of the defect site. Moreover, serum markers and histological evaluation were assessed to determine osseous tissue regeneration and turnover. Live CT and microCT indicated cBG implants displayed expedited bone repair vs, BG alone, already at 6 weeks post defect induction. cBG also displayed a shorter distance between the defect edges and greater mineral apposition distance compared to BG. Summerizing, the data support the addition of chondrocytes to bone substitute, accelerates the formation of new bone within a critical size defect.

Effectiveness of Apigenin, Resveratrol, and Curcumin as Adjuvant Nutraceuticals for Calvarial Bone Defect Healing: An In Vitro and Histological Study on Rats

Bone healing is a major clinical issue, especially in bone defects of critical dimensions. Some studies have reported in vivo positive effects on bone healing by some bioactive compounds, such as the phenolic derivatives found in vegetables and plants, such as resveratrol, curcumin, and apigenin. The aim of this work was (1) to analyze in vitro in human dental pulp stem cells the effects of these three natural compounds on the gene expression of related genes downstream to RUNX2 and SMAD5, key factor transcriptions associated with osteoblast differentiation, in order to better understand the positive effects that can occur in vivo in bone healing, and (2) to evaluate in vivo the effects on bone healing of critical-size defects in the calvaria in rats of these three nutraceuticals tested in parallel and for the first time administered by the gastric route. Upregulation of the RUNX2, SMAD5, COLL1, COLL4, and COLL5 genes in the presence of apigenin, curcumin, and resveratrol was detected. In vivo, apigenin induced more consistent significant bone healing in critical-size defects in rat calvaria compared to the other study groups. The study findings encourage a possible therapeutic supplementation with nutraceuticals during the bone regeneration process.

Engineering 3D Printed Bioceramic Scaffolds to Reconstruct Critical-Sized Calvaria Defects in a Skeletally Immature Pig Model.

Three-dimensional printed bioceramic scaffolds composed of 100% β-tricalcium phosphate augmented with dipyridamole (3DPBC-DIPY) can regenerate bone across critically sized defects in skeletally mature and immature animal models. Before human application, safe and effective bone formation should be demonstrated in a large translational animal model. This study evaluated the ability of 3DPBC-DIPY scaffolds to restore critically sized calvarial defects in a skeletally immature, growing minipig. Unilateral calvarial defects (~1.4 cm) were created in 6-week-old Göttingen minipigs ( n = 12). Four defects were filled with a 1000 μm 3DPBC-DIPY scaffold with a cap (a solid barrier on the ectocortical side of the scaffold to prevent soft-tissue infiltration), four defects were filled with a 1000 μm 3DPBC-DIPY scaffold without a cap, and four defects served as negative controls (no scaffold). Animals were euthanized 12 weeks postoperatively. Calvariae were subjected to micro-computed tomography, 3D reconstruction with volumetric analysis, qualitative histologic analysis, and nanoindentation. Scaffold-induced bone growth was statistically greater than in negative controls ( P ≤ 0.001), and the scaffolds with caps produced significantly more bone generation compared with the scaffolds without caps ( P ≤ 0.001). Histologic analysis revealed woven and lamellar bone with haversian canals throughout the regenerated bone. Cranial sutures were observed to be patent, and there was no evidence of ectopic bone formation or excess inflammatory response. Reduced elastic modulus and hardness of scaffold-regenerated bone were found to be statistically equivalent to native bone ( P = 0.148 for reduced elastic modulus of scaffolds with and without caps and P = 0.228 and P = 0.902 for hardness of scaffolds with and without caps, respectively). 3DPBC-DIPY scaffolds have the capacity to regenerate bone across critically sized calvarial defects in a skeletally immature translational pig model. This study assessed the bone generative capacity of 3D-printed bioceramic scaffolds composed of 100% β-tricalcium phosphate and augmented with dipyridamole placed within critical-sized calvarial defects in a growing porcine model.

Self-assembling peptide-laden electrospun scaffolds for guided mineralized tissue regeneration.

Electrospun scaffolds are a versatile biomaterial platform to mimic fibrillar structure of native tissues extracellular matrix, and facilitate the incorporation of biomolecules for regenerative therapies. Self-assembling peptide P11-4 has emerged as a promising strategy to induce mineralization; however, P11-4 application has been mostly addressed for early caries lesions repair on dental enamel. Here, to investigate P11-4's efficacy on bone regeneration, polymeric electrospun scaffolds were developed, and then distinct concentrations of P11-4 were physically adsorbed on the scaffolds. P11-4-laden and pristine (P11-4-free) electrospun scaffolds were immersed in simulated body fluid and mineral precipitation identified by SEM. Functional groups and crystalline phases were analyzed by FTIR and XRD, respectively. Cytocompatibility, mineralization, and gene expression assays were conducted using stem cells from human exfoliated deciduous teeth. To investigate P11-4-laden scaffolds potential to induce in vivo mineralization, an established rat calvaria critical-size defect model was used. We successfully synthesized nanofibrous (∼ 500nm fiber diameter) scaffolds and observed that functionalization with P11-4 did not affect the fibers' diameter. SEM images indicated mineral precipitation, while FTIR and XRD confirmed apatite-like formation and crystallization for P11-4-laden scaffolds. In addition, P11-4-laden scaffolds were cytocompatible, highly stimulated cell-mediated mineral deposition, and upregulated the expression of mineralization-related genes compared to pristine scaffolds. P11-4-laden scaffolds led to enhanced in vivo bone regeneration after 8 weeks compared to pristine PCL. Electrospun scaffolds functionalized with P11-4 are a promising strategy for inducing mineralized tissues regeneration in the craniomaxillofacial complex.

Potential Use of 3D CORAGRAF-Loaded PDGF-BB in PLGA Microsphere Seeded Mesenchymal Stromal Cells in Enhancing the Repair of Calvaria Critical-Size Bone Defect in Rat Model.

Our previous study evidenced that the 3D CORAGRAF loaded with PLGA microsphere constitutes PDGF-BB can support cell attachment and proliferation and can induce an osteogenic commitment of mesenchymal stromal cells in the in vitro condition. However, how this construct can perform in pathophysiological conditions in terms of repairing critical bone defects is yet to be understood. A study was therefore conducted to investigate the regeneration potential of calvaria critical-size defects using CORAGRAF + PLGA with PDGF-BB + mesenchymal stromal cells (MSCs) in a rat model. A 5 mm critical bone defect was created on calvaria of 40 male Sprague-Dawley rats. CORAGRAF incorporated either with or without PDGF-BB and seeded with rat bone-marrow-derived MSCs was implanted at the defect region. The bone regeneration potential of implanted constructs was assessed using micro-CT imaging and histological staining in weeks 4 and 8. The micro-CT images indicated a significant closure of defects in the cranial bone of the rats treated with 3D CORAGRAF + PLGA with PDGF-BB + MSCs on week 4 and 8 post-implantation. This finding, further supported with the histology outcome where the rat cranial defect treated with CORAGRAF + PLGA with PDGF-BB + MSCs indicated neo-bony ingrowth with organized and mature bone-like morphology as compared with other groups. The previous in vitro results substantiated with our pre-clinical findings demonstrate that the combination of CORAGRAF + PLGA with PDGF-BB + MSCs could be an ideal construct to support bone regeneration in critical bone defects. Hence, this construct can be further investigated for its safety and efficacy in large animal models, or it can be skipped to human trial prior for commercialization.

Comparison of rhBMP-2 in Combination with Different Biomaterials for Regeneration in Rat Calvaria Critical-Size Defects.

Regeneration of critical bone defects requires the use of biomaterials. The incorporation of osteoinductive agents, such as bone morphogenetic proteins (BMPs), improves bone formation. This study aimed to compare the efficacy of rhBMP-2 in combination with different materials for bone regeneration in critical-sized rat calvarial defects. This was an experimental animal study using 30 rats. In each rat, two 5-mm critical-size defects were made in the calvaria (60 bone defects in total) using a trephine. All rats were randomized to one of the six groups: control (C), autograft + rhBMP-2 (A), absorbable collagen sponge + rhBMP-2 (ACS), β-tricalcium phosphate + rhBMP-2 (B-TCP), bovine xenograft + rhBMP-2 (B), and hydroxyapatite + rhBMP-2 (HA). The outcome was assessed after 4 and 8 weeks using histological description and the histological bone healing scale. Statistical analysis was performed using the Kruskal-Wallis and Mann–Whitney U tests, with a p-value set at 0.05. The average bone healing scores per group were as follows: C group, 12.5; A group, 26.5; ACS group, 18.8; B-TCP group, 26.2; HA group, 20.9; and B group, 20.9. The C group showed a significant difference between weeks 4 and 8 (p = 0.032). Among the 4-week groups, the C group showed a significant difference compared to A (p = 0.001), ACS (p = 0.017), and B-TCP (p = 0.005) groups. The 8-week experimental group did not show any significant differences between the groups. The 5-mm critical size defect in rat calvaria requires the use of bone biomaterials to heal at 4 and 8 weeks. rhBMP-2, as applied in this study, showed no difference in new bone formation when combined with bovine, B-TCP, or HA biomaterials.

Calvaria Critical Size Defects Regeneration Using Collagen Membranes to Assess the Osteopromotive Principle: An Animal Study.

Guided bone regeneration (GBR) is a common practice in implantology, and it is necessary to use membranes in this process. The present study aimed to evaluate the osteopromotive principle of two porcine collagen membranes in critical-size defects at rats calvaria. Ninety-six Albinus Wistar rats were divided into BG (positive control), JS, CS, and CG (negative control) groups and were sacrificed at 7, 15, 30, and 60 days postoperatively. The samples were assessed by histological, histometric, immunohistochemical, and microtomographic analyses. More intense inflammatory profile was seen in the JS and CS groups (p < 0.05). At 60 days, the JS group showed a satisfactory osteopromotive behavior compared to BG (p = 0.193), while CS did not demonstrate the capacity to promote bone formation. At the immunohistochemical analysis, the CS showed mild labeling for osteocalcin (OC) and osteopontin (OP), the JS demonstrated mild to moderate for OC and OP and the BG demonstrated moderate to intense for OC and OP. The tridimensional analysis found the lowest average for the total volume of newly formed bone in the CS (84,901 mm2), compared to the BG (319,834 mm2) (p < 0.05). We conclude that the different thicknesses and treatment techniques of each membrane may interfere with its biological behavior.

In vitro and in vivo characterization of a novel tricalcium silicate-based ink for bone regeneration using laser-assisted bioprinting

Grafts aside, current strategies employed to overcome bone loss still fail to reproduce native tissue physiology. Among the emerging bioprinting strategies, laser-assisted bioprinting (LAB) offers very high resolution, allowing designing micrometric patterns in a contactless manner, providing a reproducible tool to test ink formulation. To this date, no LAB associated ink succeeded to provide a reproducible ad integrum bone regeneration on a murine calvaria critical size defect model. Using the Conformité Européenne (CE) approved BioRoot RCS® as a mineral addition to a collagen-enriched ink compatible with LAB, the present study describes the process of the development of a solidifying tricalcium silicate-based ink as a new bone repair promoting substrates in a LAB model. This ink formulation was mechanically characterized by rheology to adjust it for LAB. Printed aside stromal cells from apical papilla (SCAPs), this ink demonstrated a great cytocompatibility, with significant in vitro positive impact upon cell motility, and an early osteogenic differentiation response in the absence of another stimulus. Results indicated that the in vivo application of this new ink formulation to regenerate critical size bone defect tends to promote the formation of bone volume fraction without affecting the vascularization of the neo-formed tissue. The use of LAB techniques with this ink failed to demonstrate a complete bone repair, whether SCAPs were printed or not of at its direct proximity. The relevance of the properties of this specific ink formulation would therefore rely on the quantity applied in situ as a defect filler rather than its cell modulation properties observed in vitro. For the first time, a tricalcium silicate-based printed ink, based on rheological analysis, was characterized in vitro and in vivo, giving valuable information to reach complete bone regeneration through formulation updates. This LAB-based process could be generalized to normalize the characterization of candidate ink for bone regeneration.

Angio-osteogenic capacity of octacalcium phosphate co-precipitated with copper gluconate in rat calvaria critical-sized defect

ABSTRACT The objective of this study is to investigate the effects of octacalcium phosphate (OCP)-induced bone regeneration on angiogenesis regulated by the inclusion of copper ions in OCP in vitro and in vivo. Calcium (Ca)-deficient Cu-OCPs, containing 0.01 wt% Cu (low-Cu-OCP) and 0.12 wt% Cu (high-Cu-OCP), were synthesized with co7pper gluconate salt. The lattice parameters of Cu-OCPs tended to decrease slightly with Cu inclusion, as estimated by Rietveld analysis. Cu ions were released in OCP when the materials were incubated in the medium for human umbilical vein endothelial cells (HUVECs). The solubility of Cu-OCPs, estimated by the degree of supersaturation, was slightly higher than that of the original OCP. Cu-OCP tended to hydrolyze to an apatite structure while maintaining the crystal plate-like morphology when incubated with mesenchymal stem D1 cells in osteogenic media for 14 days. The specimens were characterized by selected area electron diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. Low-Cu-OCP significantly enhanced the HUVEC capillary cross-linking density. D1 cell differentiation was inhibited with the inclusion of Cu, even at low concentrations. The composite of low-Cu-OCP with a gelatin sponge (low-Cu-OCP/Gel) significantly enhanced angiogenesis coupled with bone regeneration when implanted in a rat calvarial critical-sized defect for 4 weeks, compared with the corresponding amount of Cu-containing Gel (Cu/Gel) or OCP/Gel materials through angiography and tissue histomorphometry. These results support the proposition that angiogenesis stimulated by low-Cu-OCP is closely related with enhanced bone regeneration.

Human oral mucosa-derived neural crest-like stem cells differentiate into functional osteoprogenitors that contribute to regeneration of critical size calvaria defects.

Regeneration of large bony defects is an unmet medical need. The therapeutic effect of fully developed bony constructs engineered in vitro from mineralized scaffold and adult stem cells is hampered by deficient long-term graft integration. The purpose of the present study was to investigate the regenerative capacity of a bony primordial construct consisting of human oral mucosa stem cells (hOMSC)-derived osteoprogenitors and absorbable Gelfoam® sponges. Gingiva and alveolar mucosa-derived hOMSC were differentiated into osteoprogenitors (Runx2 and osterix positive) and loaded into Gelfoam® sponges to generate primordial hOMSC constructs. These were implanted into critical size calvaria defects in the rat. Defects treated with human dermal fibroblasts (HDF) constructs; Gelfoam® sponges and untreated defects served as controls. After 120-day post-implantation defects treated with hOMSC constructs, HDF constructs and gelatin and untreated defects exhibited 86%, 30%, 21%, and 9% of new bone formation, respectively. Immunofluorescence analysis for human nuclear antigen (HNA), bone sialoprotein (BSP), and osteocalcin (OCN) revealed viable hOMSC-derived osteoblasts and osteocytes that formed most of the cell population of the newly formed bone at 30 and 120days post surgery. Few HNA-positive HDF that were negative for BSP and OCN were identified together with inflammatory cells in the soft tissue adjacent to new bone formation only at 30days post implantation. Collectively, the results demonstrate that primordial in vitro engineered constructs consisting of hOMSC-derived osteoprogenitors and absorbable gelatin almost completely regenerate critical size defects in an immunocompetent xenogeneic animal by differentiating into functional osteoblasts that retain the immunomodulatory ability of naïve hOMSC.

Mutual chemical effect of autograft and octacalcium phosphate implantation on enhancing intramembranous bone regeneration

ABSTRACT This study examined the effect of a mixture of octacalcium phosphate (OCP) and autologous bone on bone regeneration in rat calvaria critical-sized defect (CSD). Mechanically mixed OCP and autologous bone granules (OCP+Auto), approximately 500 to 1000 μm in diameter, and each individual material were implanted in rat CSD for 8 weeks, and subjected to X-ray micro-computed tomography (micro-CT), histology, tartrate-resistant acid phosphatase (TRAP) staining, and histomorphometry for bone regeneration. Osteoblastic differentiation from mesenchymal stem cells (D1 cells) was examined in the presence of non-contacting materials by alkaline phosphatase (ALP) activity for 21 days. The material properties and medium composition before and after the incubation were determined by selected area electron diffraction (SAED) under transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), and chemical analysis. The results showed that while bone formation coupled with TRAP-positive osteoclastic resorption and cellular ALP activity were the highest in the Auto group, a positive effect per OCP weight or per autologous bone weight on ALP activity was found. Although the OCP structure was maintained even after the incubation (SAED), micro-deposits were grown on OCP surfaces (TEM). Fibrous tissue was also exposed on the autologous bone surfaces (SEM). Through FT-IR absorption, it was determined that bone mineral-like characteristics of the phosphate group increased in the OCP + Auto group. These findings were interpreted as a structural change from OCP to the apatitic phase, a conclusion supported by the medium degree of saturation changes. The results demonstrate the mutual chemical effect of mixing OCP with autologous bone as an active bone substitute material.

In Vivo Study on Bone Response to 3D-Printed Constructs Designed from Microtomographic Images

The design of scaffolds that could adjust and adapt greatly to bone defects is a significant challenge to bone tissue engineering. Recently, 3D printing technology emerged as a process that could precisely control the architecture and design an exact geometry of the scaffold to the site or defect where it will be implanted. Thus, this research aimed to design and synthesize individualized constructs of polylactic acid (PLA) by 3D printing using microtomographic images to fit the edge of Wistar rat calvaria's critical size defects. The 3D-printed construct using the DICOM data of microtomographic images process to STL manipulated and designed by various software showed an excellent geometry. The in vitro biocompatibility assay of the 3D printing scaffold was evaluated by WST-1, showing an excellent biological response. Moreover, the in vivo evaluation of the bone regeneration process onto the rat calvaria defect model measured by the bone mineral density (BMD) at 8, 30, 60, and 90 days via micro-CT showed that at the end of the evaluation period, the 3D construct was integrated into the edges of the bone tissue, and new tissue deposited was in the process of mineralization. These findings suggest that the 3D construct matches the calvaria defect, allowing the novo mineral tissue to form. These individualized printed scaffolds may be a promising candidate in bone tissue engineering for future regeneration strategies.

Association of hyaluronic acid with a deproteinized bovine graft improves bone repair and increases bone formation in critical-size bone defects.

This study is designed to evaluate the potential of different formulations of hyaluronic acid (HA) to improve new bone formation in critical-size calvaria defect (CSD) when combined with a deproteinized bovine graft (DBG) material. Thirty male rats were used. A 5-mm-diameter CSD was created and three experimental groups (n=10) were randomly assigned based on the treatments performed. Group DBG: CSD filled with a DBG; group DBG/LV: CSD filled by the combination of DBG and HA in a low-viscosity crosslinking agent; group DBG/HV: CSD filled by the combination of DBG and HA in a high-viscosity crosslinking agent. Animals were euthanized 30 days postoperatively. Histological, histometric (percentage of newly formed bone [PNFB], percentage of remaining graft particles, histochemical, and immunohistochemical (bone morphogenetic protein 2/4 [BMP2/4], osteocalcin [OCN], and tartrate-resistant acid phosphatase [TRAP]) analyses were performed. The highest PNFB was observed in DBG/HV when compared with the other groups (P ≤0.05). DBG/LV and DBG/HV presented almost no inflammatory cells. In contrast, inflammation was observed in group DBG. Extensive resorption of graft particles was observed in group DBG, which was not present in DBG/LV and DBG/HV as confirmed by the larger size of the particles (P ≤0.05). BMP2/4 and OCN immunolabeling were higher in DBG/HV when compared with group DBG (P ≤0.05). Increased number of TRAP-positive cells was observed in DBG/LV and DBG/HV (P ≤0.05). Lower percentage of mature collagen fibers was observed in DBG/HV (P ≤0.05). The combination of HA in a high-viscosity crosslinking agent with DBG improves the bone repair process and increases the amount of newly formed bone towards CSDs in rat calvaria.

Nano-Silicate-Reinforced and SDF-1α-Loaded Gelatin-Methacryloyl Hydrogel for Bone Tissue Engineering.

PurposeAutologous bone grafts are the gold standard for treating bone defects. However, limited bone supply and morbidity at the donor site restrict its extensive use. Therefore, developing bone graft materials as an alternative to autologous grafts has gained considerable attention. Injectable hydrogels endowed with osteogenic potential have the ability to fill irregular bone defects using minimally invasive procedures and have thus been attracting researchers’ attention. However, from a clinical perspective, most fabrication methods employed for the current injectable osteogenic hydrogels are difficult and inconvenient. In the current study, we fabricated an injectable osteogenic hydrogel using a simple and convenient strategy.Materials and MethodsGelatin-methacryloyl (GelMA) pre-polymer was synthetized. Nano silicate (SN) and stromal cell-derived factor-1 alpha (SDF-1α) were introduced into the pre-polymer to achieve injectability, controlled release property, excellent osteogenic ability, and efficient stem cell homing.ResultsThe GelMA-SN-SDF-1α demonstrated excellent injectability via a 17-G needle at room temperature. The loaded SDF-1α exhibited a long-term controlled release pattern and efficiently stimulated MSC migration and homing. The GelMA-SN-SDF-1α hydrogel amplified cell spreading, migration, osteogenic-related biomarker expression, and matrix mineralization. The GelMA-SN-SDF-1α hydrogel filled critical-sized calvaria defects in rats and demonstrated excellent bone regeneration ability, as assessed using micro-CT scanning and histomorphometric staining.ConclusionThe GelMA-SN-SDF-1α hydrogel provides a simple and convenient strategy for the fabrication of injectable osteogenic graft materials.

In vivo evaluation of interactions between biphasic calcium phosphate (BCP)-niobium pentoxide (Nb2O5) nanocomposite and tissues using a rat critical-size calvarial defect model.

Natural or synthetic biomaterials are increasingly being used to support bone tissue repair or substitution. The combination of natural calcium phosphates with biocompatible alloys is an important route towards the development of new biomaterials with bioperformance and mechanical responses to mimic those of human bones. This article evaluated the structural, physical, mechanical and biological properties of a new mechanical improved nanocomposite elaborated by association of fish biphasic calcium phosphate (BCP) and niobium pentoxide (Nb2O5). The nanocomposite (Nb-BCP) and the pure BCP, used as a positive control, were obtained by powder metallurgy. The density, porosity and microhardness were measured. The structural analysis was determined by X-ray diffraction (XRD) and the biological properties were studied in histological sections of critical size calvaria defects in rats, 7, 15, 30, 45 and 60 days after implantation of disks of both materials. Morphological description was made after scanning electron microscopy (SEM) and optical microscopy analysis. After sintering, the Nb-BCP nanocomposite presented four crystalline phases: 34.36% calcium niobate (CaNb2O6), 21.68% phosphorus niobium oxide (PNb9O25), 42.55% β-tricalcium phosphate (Ca3(PO4)2) and 1.31% of niobium pentoxide (Nb2O5) and exhibited increases of 17% in density, 66% in Vickers microhardness and 180% in compressive strength compared to pure BCP. In vivo study, showed biocompatibility, bioactivity and osteoconductivity similar to pure BCP. SEM showed the formation of globular accretions over the implanted nanocomposites, representing one of the stages of bone mineralization. In conclusion, the BCP and Nb2O5 formed a nanocomposite exhibiting characteristics that are desirable for a biomaterial, such as bioperformance, higher β-TCP percentage and improved physical and mechanical properties compared to pure BCP. These characteristics demonstrate the promise of this material for supporting bone regeneration.

Calvaria critical-size defects in rats using piezoelectric equipment: a comparison with the classic trephine

Calvarial critical-size defects in rats are used to study regeneration of both craniofacial bone and long-bones. For decades, the trephine technique has been used with no notable refinements in the procedure. The use of piezoelectric surgical equipment has increased in human clinical oral and maxillofacial surgery, neurosurgery, traumatology, and orthopedics, because the devices are easy to handle, and can cut bone without damaging sensitive soft tissues such as blood vessels, nerves, and membranes. This study evaluated and compared the surgical technique and bone regeneration process between a traditional hand-drill trephine and piezoelectric equipment in a critical-size calvaria defect in a rat model. Thirty SD male rats were randomly divided into two groups and had either a 7.9mm diameter circular defect created with trephine or a 7.0mm square defect using the piezoelectric device, both creating 49 mm2 defect areas. MicroCT and histology were performed at 45 and 75d after surgery. While trephine surgeries were performed faster than piezoelectric (25.5 minutes vs 38.5 minutes), the rate of complications was much higher, with 36% of trephine rats taking 20 minutes to achieve hemostasis. Although the extent of new bone formation was similar between the two surgical groups, the piezoelectric technique resulted in 50% less variability. No additional new bone formation was observed from 45 to 75d in both techniques. Piezoelectric technique represents a refined and more reproducible technique for calvarial defect generation in comparison to classic trephine methods.