Regeneration Of Large Bone Defects Research Articles

LPS‑mediated adaptation accelerates ecto‑MSCs differentiation into osteoblasts.

Addressing the repair and regeneration of large bone defects poses significant challenges in bone tissue engineering. Despite the abundant evidence demonstrating the positive role of MSCs in osteogenesis, their limited osteogenic differentiation ability still needs to be improved. The present study used lipopolysaccharide (LPS) to enhance the osteogenic properties of ecto‑mesenchymal stem cells (EMSCs). Human nasal respiratory mucosa‑derived EMSCs were cultured on plates and stimulated with LPS for 5 days prior to undergoing osteogenic differentiation. The findings revealed that LPS effectively stimulated the osteogenic differentiation capacity of EMSCs, as evidenced by heightened alkaline phosphatase activity, elevated expression levels of osteogenic‑related proteins and enhanced mineralization of EMSCs. The present study also demonstrated that the augmentation occurred due to increased IL‑10 levels, although it was not solely attributable to this factor. Together, the findings illustrated that the LPS‑mediated adaptation of EMSCs is an active process driving osteogenic differentiation and could be a novel strategy for bone regeneration.

Biomimetic nanocomposite cryogel with enhanced peptide binding promotes therapeutic angiogenesis and bone regeneration

The effective regeneration of large bone defects faces a significant hurdle, primarily due to inadequate vascularization. This study explores the potential of enhancing vascularization and bone regeneration by incorporating whitlockite nanoparticles (nWH; Ca18Mg2(HPO4)2(PO4)12) and WKYMVm (W) peptide into a gelatin-sulfated hyaluronic acid (WH10-W) cryogel. The developed cryogels showed an average pore size of 70 μm. Further, they were characterized for degradation rate, swelling ratio, mechanical and rheological properties. The WH10-W group exhibited sustained release of the W peptide, in contrast to the rapid release observed from gelatin-hyaluronic acid. In silico binding studies revealed that the binding of the W peptide was dependent on the sulfation degree in hyaluronic acid. The prepared cryogels were cytocompatible. The combinatorial group (WH10-W) demonstrated superior pro-angiogenic compared to cryogel with only nWH or W peptide, as seen from tube formation assay, scratch test and gene expression studies. In addition, WH10-W enhanced the osteogenic differentiation as seen from the osteogenic gene expression, ALP and alizarin red s staining. Finally, mouse hind limb ischemia model demonstrated enhanced angiogenic potential, while a mouse cranial defect study showed increased new bone formation in the WH10-W group compared to all other groups. These findings suggest a promising strategy for addressing large non-load bearing bone defects.

Characterization and biological evaluation of 3D printed composite ink consisting of collagen, hyaluronic acid and calcium phosphate for bone regeneration

In large bone defects the self-healing capacity is insufficient, and the current standard treatment, autologous bone grafting, has severe disadvantages such as limited availability and donor site morbidity. Alternatively, clinically available bone graft substitutes lack spatial control over scaffold architecture to anatomically match complicated bone defects. Therefore, the aim in this study was to develop a 3D printable composite biomaterial-ink to promote healing of large bone defects. The composite biomaterial-ink consisted of an organic biopolymer matrix with tyramine modified hyaluronic acid (THA) and collagen type I (Col) mixed with osteoinductive calcium phosphate particles (CaP). The biopolymer was combined with 0, 10, 20 and 30 % of either 45–63 µm or 45–106 µm CaP. µCT imaging showed a homogeneous distribution of CaP in the THA-Col hydrogel and all composites were 3D printable. In vitro cell activity assays revealed no indirect cytotoxicity using L929 cells and high cell cytocompatibility using human mesenchymal stromal cells (hMSCs). Additionally, all composites supported in vitro osteogenic differentiation of hMSCs. This study highlights the development of a 3D printable composite biomaterial-ink using CaP and THA-Col hydrogel that holds significant potential to be used as patient-specific bone graft substitute for the regeneration of large bone defects. Statement of significanceThis paper introduces a 3D printable composite biomaterial-ink made of osteoinductive calcium phosphate particles combined with matrix biopolymers collagen and hyaluronic acid, which was chemically modified to introduce shear thinning and shape fixation properties for 3D printing. The chemical modification only involves a small percentage of functional groups, preserving hyaluronan's biological properties. We demonstrated printability, the homogeneous distribution of the mineral phase, cytocompatibility and that the composites support osteogenesis of primary human mesenchymal stromal cells from multiple donors. The printability of the composite biomaterial-ink allows the creation of patient-specific implants with controlled geometry on porosity. This study contributes towards engineering personalized implants for replacing autologous bone grafting in all clinical situations where the bone self-healing capacity is insufficient.

A Biomimetic Fibrous Composite Scaffold with Nanotopography-Regulated Mineralization for Bone Defect Repair.

The effective regeneration of large bone defects via bone tissue engineering is challenging due to the difficulty in creating an osteogenic microenvironment. Inspired by the fibrillar architecture of the natural extracellular matrix, we developed a nanoscale bioengineering strategy to produce bone fibril-like composite scaffolds with enhanced osteogenic capability. To activate the surface for biofunctionalization, self-adaptive ridge-like nanolamellae were constructed on poly(ε-caprolactone) (PCL) electrospinning scaffolds via surface-directed epitaxial crystallization. This unique nanotopography with a markedly increased specific surface area offered abundant nucleation sites for Ca2+ recruitment, leading to a 5-fold greater deposition weight of hydroxyapatite than that of the pristine PCL scaffold under stimulated physiological conditions. Bone marrow mesenchymal stem cells (BMSCs) cultured on bone fibril-like scaffolds exhibited enhanced adhesion, proliferation, and osteogenic differentiation in vitro. In a rat calvarial defect model, the bone fibril-like scaffold significantly accelerated bone regeneration, as evidenced by micro-CT, histological histological and immunofluorescence staining. This work provides the way for recapitulating the osteogenic microenvironment in tissue-engineered scaffolds for bone repair.

Repair of Rat Calvarial Critical-Sized Defects Using Heparin-Conjugated Fibrin Hydrogel Containing BMP-2 and Adipose-Derived Pericytes.

The repair of critical-sized calvarial defects is a challenging problem for orthopedic surgery. One of the promising strategies of bone bioengineering to enhance the efficacy of large bone defect regeneration is the combined delivery of stem cells with osteoinductive factors within polymer carriers. The purpose of the research was to study the regenerative effects of heparin-conjugated fibrin (HCF) hydrogel containing bone morphogenetic protein 2 (BMP-2) and adipose-derived pericytes (ADPs) in a rat critical-sized calvarial defect model. In vitro analysis revealed that the HCF hydrogel was able to control the BMP-2 release and induce alkaline phosphatase (ALP) activity in neonatal rat osteoblasts. In addition, it was found that eluted BMP-2 significantly induced the osteogenic differentiation of ADPs. It was characterized by the increased ALP activity, osteocalcin expression and calcium deposits in ADPs. In vivo studies have shown that both HCF hydrogel with BMP-2 and HCF hydrogel with pericytes are able to significantly increase the regeneration of critical-sized calvarial defects in comparison with the control group. Nevertheless, the greatest regenerative effect was found after the co-delivery of ADPs and BMP-2 into a critical-sized calvarial defect. Thus, our findings suggest that the combined delivery of ADPs and BMP-2 in HCF hydrogel holds promise to be applied as an alternative biopolymer for the critical-sized bone defect restoration.

Collagen molecular organization preservation in human fascia lata and periosteum after tissue engineering.

Large bone defect regeneration remains a major challenge for orthopedic surgeons. Tissue engineering approaches are therefore emerging in order to overcome this limitation. However, these processes can alter some of essential native tissue properties such as intermolecular crosslinks of collagen triple helices, which are known for their essential role in tissue structure and function. We assessed the persistence of extracellular matrix (ECM) properties in human fascia lata (HFL) and periosteum (HP) after tissue engineering processes such as decellularization and sterilization. Harvested from cadaveric donors (N = 3), samples from each HFL and HP were decellularized following five different chemical protocols with and without detergents (D1-D4 and D5, respectively). D1 to D4 consisted of different combinations of Triton, Sodium dodecyl sulfate and Deoxyribonuclease, while D5 is routinely used in the institutional tissue bank. Decellularized HFL tissues were further gamma-irradiated (minimum 25kGy) in order to study the impact of sterilization on the ECM. Polarized light microscopy (PLM) was used to estimate the thickness and density of collagen fibers. Tissue hydration and content of hydroxyproline, enzymatic crosslinks, and non-enzymatic crosslinks (pentosidine) were semi-quantified with Raman spectroscopy. ELISA was also used to analyze the maintenance of the decorin (DCN), an important small leucine rich proteoglycan for fibrillogenesis. Among the decellularization protocols, detergent-free treatments tended to further disorganize HFL samples, as more thin fibers (+53.7%) and less thick ones (-32.6%) were recorded, as well as less collagen enzymatic crosslinks (-25.2%, p = 0.19) and a significant decrease of DCN (p = 0.036). GAG content was significantly reduced in both tissue types after all decellularization protocols. On the other hand, HP samples were more sensitive to the D1 detergent-based treatments, with more disrupted collagen organization and greater, though not significant loss of enzymatic crosslinks (-37.4%, p = 0.137). Irradiation of D5 HFL samples, led to a further and significant loss in the content of enzymatic crosslinks (-29.4%, p = 0.037) than what was observed with the decellularization process. Overall, the results suggest that the decellularization processes did not significantly alter the matrix. However, the addition of a gamma-irradiation is deleterious to the collagen structural integrity of the tissue.

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.

A coaxial electrospun mat coupled with piezoelectric stimulation and atorvastatin for rapid vascularized bone regeneration.

The repair of critical bone defects caused by various clinical conditions needs to be addressed urgently, and the regeneration of large bone defects depends on early vascularization. Therefore, enhanced vascularization of artificial bone grafts may be a promising strategy for the regeneration of critical-sized bone defects. Taking into account the importance of rapid angiogenesis during bone repair and the potential of piezoelectric stimulation in promoting bone regeneration, novel coaxial electrospun mats coupled with piezoelectric materials and angiogenic drugs were fabricated in this study using coaxial electrospinning technology, with a shell layer loaded with atorvastatin (AVT) and a core layer loaded with zinc oxide (ZnO). AVT was used as an angiogenesis inducer, and piezoelectric stimulation generated by the zinc oxide was used as an osteogenesis enhancer. The multifunctional mats were characterized in terms of morphology, core-shell structure, piezoelectric properties, drug release, and mechanical properties, and their osteogenic and angiogenic capabilities were validated in vivo and ex vivo. The results revealed that the coaxial electrospun mats exhibit a porous surface morphology and nanofibers with a core-shell structure, and the piezoelectricity of the mats improved with increasing ZnO content. Excellent biocompatibility, hydrophilicity and cell adhesion were observed in the multifunctional mats. Early and rapid release of AVT in the fibrous shell layer of the mat promoted angiogenesis in human umbilical vascular endothelial cells (HUVECs), whereas ZnO in the fibrous core layer harvested bioenergy and converted it into electrical energy to enhance osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs), and both modalities synergistically promoted osteogenesis and angiogenesis. Furthermore, optimal bone regeneration was achieved in a model of critical bone defects in the rat mandible. This osteogenesis-promoting effect was induced by electrical stimulation via activation of the calcium signaling pathway. This multifunctional mat coupling piezoelectric stimulation and atorvastatin promotes angiogenesis and bone regeneration, and shows great potential in the treatment of large bone defects.

Bioengineering Composite Aerogel-Based Scaffolds That Influence Porous Microstructure, Mechanical Properties and In Vivo Regeneration for Bone Tissue Application.

Tissue regeneration of large bone defects is still a clinical challenge. Bone tissue engineering employs biomimetic strategies to produce graft composite scaffolds that resemble the bone extracellular matrix to guide and promote osteogenic differentiation of the host precursor cells. Aerogel-based bone scaffold preparation methods have been increasingly improved to overcome the difficulties in balancing the need for an open highly porous and hierarchically organized microstructure with compression resistance to withstand bone physiological loads, especially in wet conditions. Moreover, these improved aerogel scaffolds have been implanted in vivo in critical bone defects, in order to test their bone regeneration potential. This review addresses recently published studies on aerogel composite (organic/inorganic)-based scaffolds, having in mind the various cutting-edge technologies and raw biomaterials used, as well as the improvements that are still a challenge in terms of their relevant properties. Finally, the lack of 3D in vitro models of bone tissue for regeneration studies is emphasized, as well as the need for further developments to overcome and minimize the requirement for studies using in vivo animal models.

Tailoring the Microarchitectures of 3D Printed Bone-like Scaffolds for Tissue Engineering Applications.

Material extrusion (MEX), commonly referred to as fused deposition modeling (FDM) or fused filament fabrication (FFF), is a versatile and cost-effective technique to fabricate suitable scaffolds for tissue engineering. Driven by a computer-aided design input, specific patterns can be easily collected in an extremely reproducible and repeatable process. Referring to possible skeletal affections, 3D-printed scaffolds can support tissue regeneration of large bone defects with complex geometries, an open major clinical challenge. In this study, polylactic acid scaffolds were printed resembling trabecular bone microarchitecture in order to deal with morphologically biomimetic features to potentially enhance the biological outcome. Three models with different pore sizes (i.e., 500, 600, and 700 µm) were prepared and evaluated by means of micro-computed tomography. The biological assessment was carried out seeding SAOS-2 cells, a bone-like cell model, on the scaffolds, which showed excellent biocompatibility, bioactivity, and osteoinductivity. The model with larger pores, characterized by improved osteoconductive properties and protein adsorption rate, was further investigated as a potential platform for bone-tissue engineering, evaluating the paracrine activity of human mesenchymal stem cells. The reported findings demonstrate that the designed microarchitecture, better mimicking the natural bone extracellular matrix, favors a greater bioactivity and can be thus regarded as an interesting option for bone-tissue engineering.

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.

Fabrication of Interleukin-4 Encapsulated Bioactive Microdroplets for Regulating Inflammation and Promoting Osteogenesis.

Despite the inherent regenerative ability of bone, large bone defect regeneration remains a major clinical challenge for orthopedic surgery. Therapeutic strategies medicated by M2 phenotypic macrophages or M2 macrophage inducer have been widely used to promote tissue remodeling. In this study, ultrasound-responsive bioactive microdroplets (MDs) encapsulated with bioactive molecule interleukin-4 (IL4, hereafter designated MDs-IL4) were fabricated to regulate macrophage polarization and potentiate the osteogenic differentiation of human mesenchymal stem cells (hBMSCs). The MTT assay, live and dead staining, and phalloidin/DAPI dual staining were used to evaluate biocompatibility in vitro. H&E staining was used to evaluate biocompatibility in vivo. Inflammatory macrophages were further induced via lipopolysaccharide (LPS) stimulation to mimic the pro-inflammatory condition. The immunoregulatory role of the MDs-IL4 was tested via macrophage phenotypic marker gene expression, pro-inflammatory cytokine level, cell morphological analysis, and immunofluorescence staining, etc. The immune-osteogenic response of hBMSCs via macrophages and hBMSCs interactions was further investigated in vitro. The bioactive MDs-IL4 scaffold showed good cytocompatibility in RAW 264.7 macrophages and hBMSCs. The results confirmed that the bioactive MDs-IL4 scaffold could reduce inflammatory phenotypic macrophages, as evidenced by changing in morphological features, reduction in pro-inflammatory marker gene expression, increase of M2 phenotypic marker genes, and inhibition of pro-inflammatory cytokine secretion. Additionally, our results indicate that the bioactive MDs-IL4 could significantly enhance the osteogenic differentiation of hBMSCs via its potential immunomodulatory properties. Our results demonstrate that the bioactive MDs-IL4 scaffold could be used as novel carrier system for other pro-osteogenic molecules, thus having potential applications in bone tissue regeneration.

Impact of Different Preparations of Tooth Graft vs Xenogeneic Bone Graft on Bone Healing: An Experimental Study.

This study aims to compare the effect of demineralized xenogeneic tooth graft in its two forms, particulate and block, with bovine xenograft in the healing of a rabbit tibial bone defect model. Two monocortical bony defects were made in the right tibias of 36 rabbits, and were divided into four groups. Group I defects were left empty, while group II, III, and IV were filled with bovine xenograft, demineralized particulate tooth graft, and demineralized perforated block tooth graft, respectively for evaluation of the bone healing process. Three rabbits from each group were euthanized at 2, 4, and 6 weeks after surgery. The bone specimens were processed and stained with hematoxylin and eosin (H&E) and osteopontin (OPN) immunohistochemical staining. The results were subjected to image analysis and quantitative evaluation. Demineralized particulate tooth graft showed the best bone healing capacity compared to all other groups at all time points tested, as it showed a large amount of the formed bone, rapid closure of the defect with a significant increase in OPN expression, and the least amount of the residual grafted particles. In comparison to bovine xenograft and demineralized dentin block graft, the demineralized particulate tooth grafting material is a promising bone grafting substitute as it proved to be osteoconductive, biocompatible, and bioresorbable. Demineralized tooth grafting material can aid in the regeneration of large bone defects, leading to improvement in the filling of the bone defects which can help in oral and maxillofacial reconstruction.

A Review of Biomimetic and Biodegradable Magnetic Scaffolds for Bone Tissue Engineering and Oncology

Bone defects characterized by limited regenerative properties are considered a priority in surgical practice, as they are associated with reduced quality of life and high costs. In bone tissue engineering, different types of scaffolds are used. These implants represent structures with well-established properties that play an important role as delivery vectors or cellular systems for cells, growth factors, bioactive molecules, chemical compounds, and drugs. The scaffold must provide a microenvironment with increased regenerative potential at the damage site. Magnetic nanoparticles are linked to an intrinsic magnetic field, and when they are incorporated into biomimetic scaffold structures, they can sustain osteoconduction, osteoinduction, and angiogenesis. Some studies have shown that combining ferromagnetic or superparamagnetic nanoparticles and external stimuli such as an electromagnetic field or laser light can enhance osteogenesis and angiogenesis and even lead to cancer cell death. These therapies are based on in vitro and in vivo studies and could be included in clinical trials for large bone defect regeneration and cancer treatments in the near future. We highlight the scaffolds' main attributes and focus on natural and synthetic polymeric biomaterials combined with magnetic nanoparticles and their production methods. Then, we underline the structural and morphological aspects of the magnetic scaffolds and their mechanical, thermal, and magnetic properties. Great attention is devoted to the magnetic field effects on bone cells, biocompatibility, and osteogenic impact of the polymeric scaffolds reinforced with magnetic nanoparticles. We explain the biological processes activated due to magnetic particles' presence and underline their possible toxic effects. We present some studies regarding animal tests and potential clinical applications of magnetic polymeric scaffolds.

Improvement of bioactivity with dual bioceramic incorporation to nanofibrous PCL scaffolds

Bone tissue injuries, diseases or related clinical interventions require bone tissue engineering (BTE) approaches for regeneration of large bone defects, especially for compromised situations. Most BTE applications in literature focused on composites of polymers with a single type of bioceramic. However, native bone matrix has various inorganic components. Accordingly, this study aimed to investigate the use of dual bioceramics in BTE scaffolds prepared by wet-electrospinning of Poly-caprolactone (PCL) and bioceramics; hydroxyapatite (HA), carbonated hydroxyapatite (CHA) and akermanite (AKR). After physicochemical characterizations of single and dual bioceramic-discs, it was found that AKR component should be at most 10% and thus, fibrous 3D composite scaffolds (PCL/HA; PCL/CHA; PCL/90HA/10AKR; PCL/90CHA/10AKR) were prepared. Scaffolds were then compared for degradation, swelling and microstructure properties. PCL/CHA, PCL/HA/AKR and PCL/CHA/AKR scaffolds improved apatite formation and presence of CHA, AKR or both in scaffolds increased Ca/P precipitation compared to HA involving scaffolds. Specifically, when two bioceramics were introduced, there was a noticeable increase in mineralization. No significant weight change was detected for scaffolds after two weeks of incubation. Presence of bioceramics in scaffolds significantly reduced swelling of PCL scaffolds with PCL/90HA10AKR and PCL/90CHA10AKR having higher swelling results than PCL/HA and PCL/CHA, respectively. Additionally, scaffolds with dual bioceramics (90HA10AKR, 90CHA10AKR) had higher cell proliferation for bone cells (Saos-2). In terms of osteogenic activity all groups showed increased ALP activity at 14 days compared to 7 days with PCL/CHA scaffolds having the highest value. Results suggests that newly developed PCL/90HA10AKR and PCL/90CHA10AKR composites hold potential for bone tissue engineering applications.

Scaffold Guided Bone Regeneration for the Treatment of Large Segmental Defects in Long Bones.

Bone generally displays a high intrinsic capacity to regenerate. Nonetheless, large osseous defects sometimes fail to heal. The treatment of such large segmental defects still represents a considerable clinical challenge. The regeneration of large bone defects often proves difficult, since it relies on the formation of large amounts of bone within an environment impedimental to osteogenesis, characterized by soft tissue damage and hampered vascularization. Consequently, research efforts have concentrated on tissue engineering and regenerative medical strategies to resolve this multifaceted challenge. In this review, we summarize, critically evaluate, and discuss present approaches in light of their clinical relevance; we also present future advanced techniques for bone tissue engineering, outlining the steps to realize for their translation from bench to bedside. The discussion includes the physiology of bone healing, requirements and properties of natural and synthetic biomaterials for bone reconstruction, their use in conjunction with cellular components and suitable growth factors, and strategies to improve vascularization and the translation of these regenerative concepts to in vivo applications. We conclude that the ideal all-purpose material for scaffold-guided bone regeneration is currently not available. It seems that a variety of different solutions will be employed, according to the clinical treatment necessary.

Scaffolds with high oxygen content support osteogenic cell survival under hypoxia.

Regeneration of large bone defects is a significant clinical challenge with variable success, but tissue engineering strategies are promising for rapid and effective bone regeneration. Maintaining an adequate oxygen level within implanted scaffolds is a major obstacle in bone tissue engineering. We developed a new oxygen-generating scaffold by electrospinning polycaprolactone with calcium peroxide (CaO2) nanocuboids (CPNCs) and characterized the physical, chemical, and biological properties of the resulting composites. Our scaffolds are highly porous and composed of submicron fibers that include CPNC as confirmed with XRD and FTIR analyses. Scaffolds containing CPNC provided controlled oxygen release for 14-days and supported cell proliferation while protecting preosteoblasts from hypoxia-induced cell death. Oxygen-generating scaffolds also facilitated bone mimetic defect contraction in vitro. The results suggest that our approach can be used to develop tissue-engineered products which target bone defects.

3D bioprinting of cartilaginous templates for large bone defect healing.

Damaged or diseased bone can be treated using autografts or a range of different bone grafting biomaterials, however limitations with such approaches has motivated increased interest in developmentally inspired bone tissue engineering (BTE) strategies that seek to recapitulate the process of endochondral ossification (EO) as a means of regenerating critically sized defects. The clinical translation of such strategies will require the engineering of scaled-up, geometrically defined hypertrophic cartilage grafts that can be rapidly vascularised and remodelled into bone in mechanically challenging defect environments. The goal of this study was to 3D bioprint mechanically reinforced cartilaginous templates and to assess their capacity to regenerate critically sized femoral bone defects. Human mesenchymal stem/stromal cells (hMSCs) were incorporated into fibrin based bioinks and bioprinted into polycaprolactone (PCL) frameworks to produce mechanically reinforced constructs. Chondrogenic priming of such hMSC laden constructs was required to support robust vascularisation and graft mineralisation in vivo following their subcutaneous implantation into nude mice. With a view towards maximising their potential to support endochondral bone regeneration, we next explored different in vitro culture regimes to produce chondrogenic and early hypertrophic engineered grafts. Following their implantation into femoral bone defects within transiently immunosuppressed rats, such bioprinted constructs were rapidly remodelled into bone in vivo, with early hypertrophic constructs supporting higher levels of vascularisation and bone formation compared to the chondrogenic constructs. Such early hypertrophic bioprinted constructs also supported higher levels of vascularisation and spatially distinct patterns of new formation compared to BMP-2 loaded collagen scaffolds (here used as a positive control). In conclusion, this study demonstrates that fibrin based bioinks support chondrogenesis of hMSCs in vitro, which enables the bioprinting of mechanically reinforced hypertrophic cartilaginous templates capable of supporting large bone defect regeneration. These results support the use of 3D bioprinting as a strategy to scale-up the engineering of developmentally inspired templates for BTE. STATEMENT OF SIGNIFICANCE: Despite the promise of developmentally inspired tissue engineering strategies for bone regeneration, there are still challenges that need to be addressed to enable clinical translation. This work reports the development and assessment (in vitro and in vivo) of a 3D bioprinting strategy to engineer mechanically-reinforced cartilaginous templates for large bone defect regeneration using human MSCs. Using distinct in vitro priming protocols, it was possible to generate cartilage grafts with altered phenotypes. More hypertrophic grafts, engineered in vitro using TGF-β3 and BMP-2, supported higher levels of blood vessel infiltration and accelerated bone regeneration in vivo. This study also identifies some of the advantages and disadvantages of such endochondral bone TE strategies over the direct delivery of BMP-2 from collagen-based scaffolds.

Novel structural designs of 3D-printed osteogenic graft for rapid angiogenesis

Large bone defect regeneration has always been recognized as a challenging clinical problem due to the difficulty of revascularization. Conventional treatments exhibit certain inherent disadvantages (e.g., secondary injury, immunization, and potential infections). However, three-dimensional (3D) printing technology as an emerging field can serve as an effective approach to achieve satisfactory revascularization while making up for the above limitations. A wide variety of methods can be used to facilitate blood supply during the design of a 3D-printed scaffold. Importantly, the scaffold structure lays a foundation for the entire printing object; any method to promote angiogenesis can be effective only if it is based on well-designed scaffolds. In this review, different designs related to angiogenesis are summarized by collecting the literature from recent years. The 3D-printed scaffolds are classified into four major categories and discussed in detail, from elementary porous scaffolds to the most advanced bone-like scaffolds. Finally, structural design suggestions to achieve rapid angiogenesis are proposed by analyzing the above architectures. This review can provide a reference for organizations or individual academics to achieve improved bone defect repair and regeneration using 3D printing.

Scaffold microarchitecture regulates angiogenesis and the regeneration of large bone defects

Emerging 3D printing technologies can provide exquisite control over the external shape and internal architecture of scaffolds and tissue engineering (TE) constructs, enabling systematic studies to explore how geometric design features influence the regenerative process. Here we used fused deposition modelling (FDM) and melt electrowriting (MEW) to investigate how scaffold microarchitecture influences the healing of large bone defects. FDM was used to fabricate scaffolds with relatively large fibre diameters and low porosities, while MEW was used to fabricate scaffolds with smaller fibre diameters and higher porosities, with both scaffolds being designed to have comparable surface areas. Scaffold microarchitecture significantly influenced the healing response following implantation into critically sized femoral defects in rats, with the FDM scaffolds supporting the formation of larger bone spicules through its pores, while the MEW scaffolds supported the formation of a more round bone front during healing. After 12 weeks in vivo, both MEW and FDM scaffolds supported significantly higher levels of defect vascularisation compared to empty controls, while the MEW scaffolds supported higher levels of new bone formation. Somewhat surprisingly, this superior healing in the MEW group did not correlate with higher levels of angiogenesis, with the FDM scaffold supporting greater total vessel formation and the formation of larger vessels, while the MEW scaffold promoted the formation of a dense microvasculature with minimal evidence of larger vessels infiltrating the defect region. To conclude, the small fibre diameter, high porosity and high specific surface area of the MEW scaffold proved beneficial for osteogenesis and bone regeneration, demonstrating that changes in scaffold architecture enabled by this additive manufacturing technique can dramatically modulate angiogenesis and tissue regeneration without the need for complex exogenous growth factors. These results provide a valuable insight into the importance of 3D printed scaffold architecture when developing new bone TE strategies.