Bone Regeneration In Critical-sized Defects Research Articles

Effect of a synthetic hydroxyapatite-based bone grafting material compared to established bone substitute materials on regeneration of critical-size bone defects in the ovine scapula

Abstract Lately, the potential risk of disease transmission due to the use of bovine-derived bone substitutes has become obvious, demonstrating the urgent need for a synthetic grafting material with comparable bioactive behaviour and properties. Therefore, the effect of a synthetic hydroxyapatite (HA) (Osbone®) bone grafting material on bone regeneration was evaluated 2 weeks, 1 month, and 3, 6, 12, and 18 months after implantation in critical-size bone defects in the ovine scapula and compared to that of a bovine-derived HA (Bio-Oss®) and β-tricalcium phosphate (TCP) (Cerasorb® M). New bone formation and the biodegradability of the bone substitutes were assessed histomorphometrically. Hard tissue histology and immunohistochemical analysis were employed to characterize collagen type I, alkaline phosphatase, osteocalcin, as well as bone sialoprotein expression in the various cell and matrix components of the bone tissue to evaluate the bioactive properties of the bone grafting materials. No inflammatory tissue response was detected with any the bone substitute materials studied. After 3 and 6 months, β-TCP (Cerasorb® M) showed superior bone formation when compared to both HA-based materials (3 months: β-TCP 55.65 ± 2.03% vs SHA 49.05 ± 3.84% and BHA 47.59 ± 1.97%; p ≤ 0.03; 6 months: β-TCP 62.03 ± 1.58%; SHA: 55.38 ± 2.59%; BHA: 53.44 ± 0.78%; p ≤ 0.04). Further, after 12 and 18 months, a similar degree of bone formation and bone–particle contact was noted for all three bone substitute materials without any significant differences. The synthetic HA supported new bone formation, osteogenic marker expression, matrix mineralization and good bone-bonding behaviour to an equal and even slightly superior degree compared to the bovine-derived HA. As a result, the synthetic HA can be regarded as a valuable alternative to the bovine-derived HA without the potential risk of disease transmission.

Optimized piezoelectric bone regeneration through inhibiting sympathetic nerve-bone interaction

Electrical cues are crucial for osteogenesis. To tackle critical-sized bone defects, piezoelectric materials non-invasively generate electrical stimuli via ultrasonic excitation, imitating natural bone's piezoelectricity. Two-dimensional piezoelectric nanomaterials, with their flexibility and cellular interactions, effectively improve osteogenic outcomes. Yet, the concurrent electrical and injury signals may overly activate sympathetic nerves, causing excessive norepinephrine release, which hinders osteogenic differentiation by targeting mesenchymal stem cells' β2-receptors. Addressing this, we developed a sophisticated drug carrier, encapsulating piezoelectric zinc sulfide (ZnS) in a polydopamine (PDA) coating with the β blocker propranolol (PRO), creating ZnS@PDA-PRO. This nanocarrier generates electrical signals and releases Zn2+ to promote osteogenic differentiation, while PRO mitigates sympathetic nerve inhibition, enhancing the regeneration of critical-sized bone defects.

Reconstructing Critical-Sized Mandibular Defects in a Rabbit Model: Enhancing Angiogenesis and Facilitating Bone Regeneration via a Cell-Loaded 3D-Printed Hydrogel-Ceramic Scaffold Application.

In this study, we propose a spatially patterned 3D-printed nanohydroxyapatite (nHA)/beta-tricalcium phosphate (β-TCP)/collagen composite scaffold incorporating human dental pulp-derived mesenchymal stem cells (hDP-MSCs) for bone regeneration in critical-sized defects. We investigated angiogenesis and osteogenesis in a rabbit critical-sized mandibular defect model treated with this engineered construct. The critical and synergistic role of collagen coating and incorporation of stem cells in the regeneration process was confirmed by including a cell-free uncoated 3D-printed nHA/β-TCP scaffold, a stem cell-loaded 3D-printed nHA/β-TCP scaffold, and a cell-free collagen-coated 3D-printed nHA/β-TCP scaffold in the experimental design, in addition to an empty defect. Posteuthanasia evaluations through X-ray analysis, histological assessments, immunohistochemistry staining, histomorphometry, and reverse transcription-polymerase chain reaction (RT-PCR) suggest the formation of substantial woven and lamellar bone in the cell-loaded collagen-coated 3D-printed nHA/β-TCP scaffolds. Histomorphometric analysis demonstrated a significant increase in osteoblasts, osteocytes, osteoclasts, bone area, and vascularization compared to that observed in the control group. Conversely, a significant decrease in fibroblasts/fibrocytes and connective tissue was observed in this group compared to that in the control group. RT-PCR indicated a significant upregulation in the expression of osteogenesis-related genes, including BMP2, ALPL, SOX9, Runx2, and SPP1. The findings suggest that the hDP-MSC-loaded 3D-printed nHA/β-TCP/collagen composite scaffold is promising for bone regeneration in critical-sized defects.

Effects of Innervation on Angiogenesis and Osteogenesis in Bone and Dental Tissue Engineering.

The repair and regeneration of critical-sized bone defects remain an urgent challenge. Bone tissue engineering represents an exciting solution for regeneration of large bone defects. Recently, the importance of innervation in tissue-engineered bone regeneration has been increasingly recognized. The cross talk between nerve and bone provides important clues for bone repair and regeneration. Furthermore, the promotion of angiogenesis by innervation can accelerate new bone formation. However, the mechanisms involved in the promotion of vascular and bone regeneration by the nervous system have not yet been established. In addition, simultaneous neurogenesis and vascularization in bone tissue engineering have not been fully investigated. This article represents the first review on the effects of innervation in enhancing angiogenesis and osteogenesis in bone and dental tissue engineering. Cutting-edge research on the effects of innervation through biomaterials on bone and dental tissue repairs is reviewed. The effects of various nerve-related factors and cells on bone regeneration are discussed. Finally, novel clinical applications of innervation for bone, dental, and craniofacial tissue regeneration are also examined.

Cell-free chitosan/silk fibroin/bioactive glass scaffolds with radial pore for in situ inductive regeneration of critical-size bone defects

Tissue-engineered is an effective method for repairing critical-size bone defects. The application of bioactive scaffold provides artificial matrix and suitable microenvironment for cell recruitment and extracellular matrix deposition, which can effectively accelerate the process of tissue regeneration. Among various scaffold properties, appropriate pore structure and distribution have been proven to play a crucial role in inducing cell infiltration differentiation and in-situ tissue regeneration. In this study, a chitosan (CS) /silk fibroin (SF) /bioactive glass (BG) composite scaffold with distinctive radially oriented pore structure was constructed. The composite scaffolds had stable physical and chemical properties, a unique pore structure of radial arrangement from the center to the periphery and excellent mechanical properties. In vitro biological studies indicated that the CS/SF/BG scaffold could promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and the expression of related genes due to the wide range of connected pore structures and released active elements. Furthermore, in vivo study showed CS/SF/BG scaffold with radial pores was more conducive to the repair of skull defects in rats with accelerated healing speed during the bone tissue remodeling process. These results demonstrated the developed CS/SF/BG scaffold would be a promising therapeutic strategy for the repair of bone defects regeneration.

Study on Synergistic Effects of Nanohydroxyapatite/High-Viscosity Carboxymethyl Cellulose Scaffolds Stimulated by LIPUS for Bone Defect Repair of Rats.

Despite the self-healing capacity of bone, the regeneration of critical-size bone defects remains a major clinical challenge. In this study, nanohydroxyapatite (nHAP)/high-viscosity carboxymethyl cellulose (hvCMC, 6500 mPa·s) scaffolds and low-intensity pulsed ultrasound (HA-LIPUS) were employed to repair bone defects. First, hvCMC was prepared from ramie fiber, and the degree of substitution (DS), purity, and content of NaCl of hvCMC samples were 0.91, 99.93, and 0.017%, respectively. Besides, toxic metal contents were below the permissible limits for pharmaceutically used materials. Our results demonstrated that the hvCMC is suitable for pharmaceutical use. Second, nHAP and hvCMC were employed to prepare scaffolds by freeze-drying. The results indicated that the scaffolds were porous, and the porosity was 35.63 ± 3.52%. Subsequently, the rats were divided into four groups (n = 8) randomly: normal control (NC), bone defect (BD), bone defect treated with nHAP/hvCMC scaffolds (HA), and bone defect treated with nHAP/hvCMC scaffolds and stimulated by LIPUS (HA-LIPUS). After drilling surgery, nHAP/hvCMC scaffolds were implanted in the defect region of HA and HA-LIPUS rats. Meanwhile, HA-LIPUS rats were treated by LIPUS (1.5 MHz, 80 mW cm-2) irradiation for 2 weeks. Compared with BD rats, the maximum load and bone mineral density of HA-LIPUS rats were increased by 20.85 and 51.97%, respectively. The gene and protein results indicated that nHAP/hvCMC scaffolds and LIPUS promoted the bone defect repair and regeneration of rats significantly by activating Wnt/β-catenin and inhibiting OPG/RANKL signaling pathways. Overall, compared with BD rats, nHAP/hvCMC scaffolds and LIPUS promoted bone defect repair significantly. Furthermore, the research results also indicated that there are synergistic effects for bone defect repair between the nHAP/hvCMC scaffolds and LIPUS.

Osteoimmunomodulatory bioinks for 3D bioprinting achieve complete regeneration of critical-sized bone defects

Regeneration the critical-sized bone defects remains a great challenge to clinical therapy due to the inflammatory microenvironment and lack of stem cells in the region of the bone defects. 3D bioprinted scaffolds based on bioactive ink and loaded active cells can promote the inflammatory microenvironment and cell viability, thereby enhancing bone regeneration. In this study, 10 % Gelatin-methacryloyl (GelMA)/5 %Sr substituted xonotlite (Sr-CSH) nanocomposite hydrogel was developed as a bioink to encapsulate bone marrow mesenchymal stem cells (BMSCs), and then constructed a biomimetic bone tissue by 3D bioprinting. The incorporation of Sr-CSH nanowires enhanced the printing accuracy and mechanical property of GelMA, and enhanced the osteogenic differentiation of BMSCs. In addition, Sr-CSH induced macrophage M2 polarization, which modulated the inflammatory microenvironment and further promoted osteogenic differentiation of BMSCs. In rat critical-sized calvarial defects model, 3D bioprinted scaffolds based on GelMA-Sr-CSH bioinks laden with BMSCs achieve complete bone repair. In summary, this study developed an osteoimmunomodulatory bioink, and 3D bioprinted scaffolds laden with stem cells may be an effective method for achieving complete regeneration of critical-sized bone defects.

Dual-phase blocks for regeneration of critical-sized bone defects

Repair of critical-size bone defects (CBD) remains a challenge in orthopedic surgery due to the restricted regenerative capacity of bone tissue. Herein, multi-functional biphasic calcium phosphate (BCP) blocks decorated with gelatin packs were developed for bone regeneration of CBDs. The gelatin packs consist of gelatin microspheres (GMSs) and an enzymatic crosslinked gelatin layer for bone morphogenetic protein-2 (BMP-2) and antibiotics delivery. The porous interior of the BCP block was filled with GMSs containing BMP-2 and tightly packed with an enzymatic crosslinked gelatin layer. The outermost part of the block was then covered with antibiotics-loaded GMSs. The BCP platform elaborately decorated for each zone improved the adhesion of osteoblasts and enabled the sequential release of antibiotics and BMP-2 and sustained release of BMP-2, realizing on-demand drug release according to the treatment stage. The programmed drug release of this platform significantly improved the regeneration of CBDs. In vivo experiments using dog's mandible defect models clearly demonstrated the clinical application potential of this BCP platform.

Methacrylated gelatin and platelet-rich plasma based hydrogels promote regeneration of critical-sized bone defects.

Physiological repair of large-sized bone defects requires instructive scaffolds with appropriate mechanical properties, biocompatibility, biodegradability, vasculogenic ability and osteo-inductivity. The objective of this study was to fabricate in situ injectable hydrogels using platelet-rich plasma (PRP)-loaded gelatin methacrylate (GM) and employ them for the regeneration of large-sized bone defects. We performed various biological assays as well as assessed the mechanical properties of GM@PRP hydrogels alongside evaluating the release kinetics of growth factors (GFs) from hydrogels. The GM@PRP hydrogels manifested sufficient mechanical properties to support the filling of the tissue defects. For biofunction assay, the GM@PRP hydrogels significantly improved cell migration and angiogenesis. Especially, transcriptome RNA sequencing of human umbilical vein endothelial cells and bone marrow-derived stem cells were performed to delineate vascularization and biomineralization abilities of GM@PRP hydrogels. The GM@PRP hydrogels were subcutaneously implanted in rats for up to 4 weeks for preliminary biocompatibility followed by their transplantation into a tibial defect model for up to 8 weeks in rats. Tibial defects treated with GM@PRP hydrogels manifested significant bone regeneration as well as angiogenesis, biomineralization, and collagen deposition. Based on the biocompatibility and biological function of GM@PRP hydrogels, a new strategy is provided for the regenerative repair of large-size bone defects.

Gait analysis: An effective tool to mechanically monitor the bone regeneration of critical-sized defects in tissue engineering applications.

Tissue engineering has emerged as an innovative approach to treat critical-size bone defects using biocompatible scaffolds, thus avoiding complex distraction surgeries or limited stock grafts. Continuous regeneration monitoring is essential in critical-size cases due to the frequent appearance of non-unions. This work evaluates the potential clinical use of gait analysis for the mechanical assessment of a tissue engineering regeneration as an alternative to the traditional and hardly conclusive manual or radiological follow-up. The 15-mm metatarsal fragment of eight female merino sheep was surgically replaced by a bioceramic scaffold stabilized with an external fixator. Gait tests were performed weekly by making the sheep walk on an instrumented gangway. The evolution of different kinematic and dynamic parameters was analyzed for all the animal's limbs, as well as asymmetries between limbs. Finally, potential correlation in the recovery of the gait parameters was evaluated through the linear regression models. After surgery, the operated limb has an altered way of carrying body weight while walking. Its loading capacity was significantly reduced as the stance phases were shorter and less impulsive. The non-operated limbs compensated for this mobility deficit. All parameters were normalizing during the consolidation phase while the bone callus was simultaneously mineralizing. The results also showed high levels of asymmetry between the operated limb and its contralateral, which exceeded 150% when analyzing the impulse after surgery. Gait recovery significantly correlated between symmetrical limbs. Gait analysis was presented as an effective, low-cost tool capable of mechanically predicting the regeneration of critical-size defects treated by tissue engineering, as comparing regeneration processes or novel scaffolds. Despite the progressive normalization as the callus mineralized, the bearing capacity reduction and the asymmetry of the operated limb were more significant than in other orthopedic alternatives.

Poly-ε-Caprolactone 3D-Printed Porous Scaffold in a Femoral Condyle Defect Model Induces Early Osteo-Regeneration.

Large bone reconstruction following trauma poses significant challenges for reconstructive surgeons, leading to a healthcare burden for health systems, long-term pain for patients, and complex disorders such as infections that are difficult to resolve. The use of bone substitutes is suboptimal for substantial bone loss, as they induce localized atrophy and are generally weak, and unable to support load. A combination of strong polycaprolactone (PCL)-based scaffolds, with an average channel size of 330 µm, enriched with 20% w/w of hydroxyapatite (HA), β-tricalcium phosphate (TCP), or Bioglass 45S5 (Bioglass), has been developed and tested for bone regeneration in a critical-size ovine femoral condyle defect model. After 6 weeks, tissue ingrowth was analyzed using X-ray computed tomography (XCT), Backscattered Electron Microscopy (BSE), and histomorphometry. At this point, all materials promoted new bone formation. Histological analysis showed no statistical difference among the different biomaterials (p > 0.05), but PCL-Bioglass scaffolds enhanced bone formation in the center of the scaffold more than the other types of materials. These materials show potential to promote bone regeneration in critical-sized defects on load-bearing sites.

Effect of systemic atorvastatin on bone regeneration in critical-sized defects in hyperlipidemia: an experimental study

PurposeHypocholesterolemic medications similar to atorvastatin are efficient in lowering blood lipid levels; however, compared to other medications in the statin family, their impact on bone metabolism is claimed to be insufficient. The impact of atorvastatin on bone regeneration in dental implantology in individuals with hyperlipidemia who received atorvastatin in the clinic is doubtful.MethodsIn the study, 16 male New Zealand rabbits of 6 months were used. All rabbits were fed a high-cholesterol diet for 8 weeks, and hyperlipidemia was created. It was confirmed that the total cholesterol level in rabbits was above 105 mg/dl. A critical-sized defect was created in the mandible. The defect was closed with xenograft and membrane. Oral 10 mg/kg atorvastatin was started in the experimental group, and no drug was administered in the control group. At 16th week, animals were sacrificed. For histomorphological examination, the new bone area, osteoclast, and osteoblast activities were evaluated.ResultsWhile new bone area (45,924 µm2, p < 0.001) and AP intensities (105.645 ± 16.727, p = 0.006) were higher in the atorvastatin group than in the control group, TRAP intensities in the control group (82.192 ± 5.346, p = 0.021) were higher than that in the atorvastatin group.ConclusionsIt has been found that high blood lipid levels will adversely affect bone graft healing and the use of systemic atorvastatin contributes to bone healing. Clinicians should pay attention to the selection of surgical materials, considering the importance of questioning drug use in their patients and the risks in cases of non-use.

Bone regeneration in critical-size defects of the mandible using biomechanically adapted CAD/CAM hybrid scaffolds: An in vivo study in miniature pigs

The study aimed to analyze bone regeneration in critical-size defects using hybrid scaffolds biomechanically adapted to the specific defect and adding the growth factor rhBMP-2.For this animal study, ten minipigs underwent bilateral defects in the corpus mandibulae and were subsequently treated with novel cylindrical hybrid scaffolds. These scaffolds were designed digitally to suit the biomechanical requirements of the mandibular defect, utilizing finite element analysis. The scaffolds comprised zirconium dioxide-tricalcium phosphate (ZrO2-TCP) support struts and TCP foam ceramics. One scaffold in each animal was loaded with rhBMP-2 (100 μg/cm³), while the other served as an unloaded negative control. Fluorescent dyes were administered every 2 weeks, and computed tomography (CT) scans were conducted every 4 weeks. Euthanasia was performed after 3 months, and samples were collected for examination using micro-CT and histological evaluation of both hard and soft tissue.Intravital CT examinations revealed minor changes in radiographic density from 4 to 12 weeks postoperatively. In the group treated with rhBMP-2, radiographic density shifted from 2513 ± 128 (mean ± SD) to 2606 ± 115 Hounsfield units (HU), while the group without rhBMP-2 showed a change from 2430 ± 131 to 2601 ± 67 HU. Prior to implantation, the radiological density of samples measured 1508 ± 30 mg HA/cm³, whereas post-mortem densities were 1346 ± 71 mg HA/cm³ in the rhBMP-2 group and 1282 ± 91 mg HA/cm³ in the control group (p = 0.045), as indicated by micro-CT measurements. The histological assessment demonstrated successful ossification in all specimens. The newly formed bone area proportion was significantly greater in the rhBMP-2 group (48 ± 10%) compared with the control group without rhBMP-2 (42 ± 9%, p = 0.03). The mean area proportion of remaining TCP foam was 23 ± 8% with rhBMP-2 and 24 ± 10% without rhBMP-2.Successful bone regeneration was accomplished by implanting hybrid scaffolds into critical-size mandibular defects. Loading these scaffolds with rhBMP-2 led to enhanced bone regeneration and a uniform distribution of new bone formation within the hybrid scaffolds. Further studies are required to determine the adaptability of hybrid scaffolds for larger and potentially segmental defects in the maxillofacial region.

A Self-Adaptive Biomimetic Periosteum Employing Nitric Oxide Release for Augmenting Angiogenesis in Bone Defect Regeneration.

The periosteum plays a vital role in the regeneration of critical-size bone defects and highly comminuted fractures, promoting the differentiation of osteoblasts, accelerating the reconstruction of the vascular network, and guiding bone tissue regeneration. However, the materials loaded with exogenous growth factors are limited by the release and activity of the elements. Therefore, the material structure must be carefully designed for the periosteal function. Here, a self-adaptive biomimetic periosteum strategy is proposed, which is a novel interpenetrating double network hydrogel consisting of diselenide-containing gelatin and calcium alginate (modified natural collagen and polysaccharide) to enhance the stability, anti-swelling, and delayed degradation of the hydrogel. The diselenide bond continuously releases nitric oxide (NO) by metabolizing endogenous nitrosated thiols (RSNO), activates the nitric oxide-cycle guanosine monophosphate (NO-cGMP) signal pathway, coordinates the coupling effect of angiogenesis and osteogenesis, and accelerates the repair of bone defects. This self-adaptive biomimetic periosteum with the interpenetrating double network structure formed by the diselenide-containing gelatin and calcium alginate has been proven to be safe and effective in repairing critical-size bone defects and is expected to provide a promising strategy for solving clinical problems.

Hierarchically Porous Implants Orchestrating a Physiological Viscoelastic and Piezoelectric Microenvironment for Bone Regeneration.

The extracellular matrix microenvironment of bone tissue comprises several physiological cues. Thus, artificial bone substitute materials with a single cue are insufficient to meet the demands for bone defect repair. Regeneration of critical-size bone defects remains challenging in orthopedic surgery. Intrinsic viscoelastic and piezoelectric cues from collagen fibers play crucial roles in accelerating bone regeneration, but scaffolds or implants providing integrated cues have seldom been reported. In this study, we aimed to design and prepare hierarchically porous poly(methylmethacrylate)/polyethyleneimine/poly(vinylidenefluoride) composite implants presenting a similar viscoelastic and piezoelectric microenvironment to bone tissue via anti-solvent vapor-induced phase separation. The viscoelastic and piezoelectric cues of the composite implants for human bone marrow mesenchymal stem cell line stimulated and activated Piezo1 proteins associated with mechanotransduction signaling pathways. Cortical and spongy bone exhibited excellent regeneration and integration in models of critical-size bone defects on the knee joint and femur in vivo. This study demonstrated that implants with integrated physiological cues were promising artificial bone substitute materials for regenerating critical-size bone defects. This article is protected by copyright. All rights reserved.

The Use of Human Sterilized Crushed Tooth Particles Compared with BTCP Biomaterial and Empty Defects in Bone Formation inside Critical Rabbit Calvaria Sites.

This study aimed to assess the bone regeneration of critical-size defects in rabbit calvaria filled with freshly crushed extracted teeth, comparing them with BTCP biomaterial and empty sites. Materials and methods: Twenty-one female New Zealand rabbits were used in this study. Two critical-size defects 6 mm in size were created in the skull bone, each with a 3 mm separation between them. Three experimental groups were evaluated: Group A (human sterilized crushed teeth granules alone), Group B (Bioner Bone, Bioner Sitemas Implantológicos), and Group C (unfilled defects). The animals were sacrificed at 4 and 8 weeks. Evaluation of the samples involved histological and histomorphometric analyses with radiographic evaluation. The histological evaluation showed a higher volume reduction in Group A compared with Group B (p < 0.05) and Control. Group A showed the highest values for cortical closure and bone formation around the particles, followed by Group B and Group C (p < 0.05). Within the limitations of this animal study, we can conclude that the use of human tooth particles leads to increased bone formation and reduced connective tissue in critical-size defects in rabbit calvaria when compared to BTCP biomaterial. The calvarial model is a robust base for the evaluation of different biomaterials.

Photobiomodulation and Inorganic Bovine Bone in Guided Bone Regeneration: Histomorphometric Analysis in Rats.

The objective of this study was to evaluate the efficacy of photobiomodulation in the bone regeneration of critical-sized defects (CSD) filled with inorganic bovine bone associated or not with collagen membranes. The study has been conducted on 40 critical defects in the calvaria of male rats, divided into four experimental groups (n = 10): (1) DBBM (deproteinized bovine bone mineral); (2) GBR (DBBM+collagen membrane); (3) DBBM+P (DBBM+photobiomodulation); and (4) GBR+P (GBR+photobiomodulation). At 30 days postoperative, the animals were euthanized, and after the tissue had been processed, histological, histometric, and statistical analyses were performed. The analyses have taken into account newly formed bone area (NBA), linear bone extension (LBE), and residual particle area (RPA) as variables. The Kruskal-Wallis test has been performed, followed by the Dwass-Steel-Critchlow-Fligner test for comparison between groups (p < 0.05). When the DBBM+P group was compared to the DBBM group, it was possible to observe significant statistical differences in all the variables analyzed (p < 0.05). The application of photobiomodulation in guided bone regeneration (GBR+P) has shown a decrease in the median value for the RPA variable (26.8) when compared to the GBR group (32.4), with a significant statistical difference; however, for NBA and LBE, the therapy has not provided significant results.

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

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

Strontium Ion-Functionalized Nano-Hydroxyapatite/Chitosan Composite Microspheres Promote Osteogenesis and Angiogenesis for Bone Regeneration

Critical-size bone defects are an important problem in clinical practice, which usually occurs in severe trauma, or tumor resection, and cannot heal completely and autonomously. Implantation of grafts is often required to promote the regeneration of critical-size bone defects. Metal ions play an important role in human health, as they affect the body's metabolism and the tissue function. Strontium ions (Sr2+) can promote osteogenesis and angiogenesis. Herein, we prepared nano-hydroxyapatite (nHA)/chitosan (CS) composite microspheres with a uniform particle size distribution and an extracellular matrix-like nanofiber structure using microfluidic technology and direct alkali-induced gelation. Strontium ions were stably added into the microspheres by using polydopamine (PDA) to chelate metal ions forming a bone repair material (nHA/CS@PDA-Sr) with good bioactivity. The coordination reaction of PDA can effectively control the release of strontium ions and avoid the negative effects caused by the high strontium concentration. Our in vitro experiments showed that the composite microspheres had good biocompatibility and that the PDA coating promotes cell adhesion. The slow release of strontium ions can effectively promote mesenchymal stem cells osteogenic differentiation and the vascularization of endothelial cells. In addition, we injected composite microspheres into cranial defects of rats to evaluate osseointegration in vivo. The results showed that nHA/CS@PDA-Sr could effectively promote bone regeneration in the defect area. This study demonstrates that composite microspheres stimulate bone repair providing a promising way for bone-defect regeneration.

The addition of zinc ions to polymer-ceramic composites accelerated osteogenic differentiation of human mesenchymal stromal cells

Critical-sized bone defects, caused by congenital disorders or trauma, are defects that will not heal spontaneously and require surgical intervention. Recent advances in biomaterial design for the treatment of such defects focus on improving their osteoinductive properties. Here, we propose a bioactive composite with high ceramic content composed of poly(ethyleneoxide terephthalate)/poly(butylene terephthalate) (1000PEOT70PBT30, PolyActive, PA) and 50 % beta-tricalcium phosphate (β-TCP) with the addition of zinc in a form of a coating on the TCP particles. Due to its essential role in bone homeostasis, we hypothesised that the addition of zinc to the polymer-ceramic composite will further enhance its osteogenic properties. β-TCP particles were immersed in a zinc solution with a concentration of 15 or 45 mM. The addition of zinc did not alter the β-TCP composition or the release of calcium or phosphate ions. 3D porous 1000PEOT70PBT30 - β-TCP scaffolds were additively manufactured by “3D fibre deposition” and their ability to support the osteogenic differentiation was assessed by culturing clinically relevant human mesenchymal stromal cells (hMSCs) on the scaffolds for 3, 7, 14 and 28 days. The expression of osteogenic gene markers was increased in the presence of both zinc concentrations. Remarkably, upregulation of osteocalcin (OCN), a late osteogenic marker, was observed after three days of culture. Furthermore, enhanced extracellular matrix (ECM) production and mineralization was observed. These findings support the existing evidence on the osteogenic properties of zinc and further demonstrate that the incorporation of zinc into a polymer-ceramic composite could be a promising strategy in the field of regeneration of critical-sized bone defects.