Bone Tissue Engineering Strategies Research Articles

Proangiogenic Hypoxia-Mimicking Agents Attenuate Osteogenic Potential of Adipose Stem/Stromal Cells

BACKGROUND:Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bone defects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelial growth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1α). This study assessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs).METHODS:Isolated AT-MSCs were characterized and treated with PHIs to assess the cellular proliferation response. Immunostaining and western-blots served to verify the HIF-1α stabilization response. The optimized concentrations for long-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiation of AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secreted phosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). In addition, stemness-related genes Kruppel-like factor 4 (KLF4), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4) were assessed.RESULTS:PHIs stabilized HIF-1α in a dose-dependent manner and showed evident dose- and time dependent antiproliferative effects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic induction on the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressed osteogenic response of AT-MSCs was accompanied by an upregulation of stemness-related genes.CONCLUSION:PHIs significantly reduced the osteogenic differentiation of AT-MSCs and rather upregulated stemness-related genes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on the osteogenic differentiation of AT-MSCs.

A novel mineralized high strength hydrogel for enhancing cell adhesion and promoting skull bone regeneration in situ

Bone defect therapy based on advanced biocomposite hydrogel scaffolds provide a promising strategy in bone tissue engineering. Inducing bone regeneration that meets the biomechanical, bioactivity and osteoconductivity criteria of bone tissue engineering is highly appealing but challenging. Here, we designed a mineralized, high-strength and tough nanocomposite hydrogel by in situ deposition of calcium phosphate hydroxide salt nanohydroxyapatite (HAP) after photopolymerization of gelatin methacryloyl (GelMA), quaternized chitosan (QCS) and functional polyhedral oligomeric silsesquioxane (POSS), in which the POSS nanoparticles served as a physical-chemical crosslinker to reinforce the hydrogel network structure and improved mineralized capacity by silicon in POSS nanoparticle bonding to calcium ions. The fabricated nanocomposite hydrogels have excellent mechanical properties (tensile strength of 328.6 ± 14.3 kPa and compressive strength of 1.71 ± 0.24 MPa) and high cytocompatibility, significantly facilitated cell adhesion and upregulated osteodifferentiation. After treatment of a rat skull defect model for 12 weeks, the scaffold of the biomineralized hydrogel remarkably promoted new bone formation and accelerated bone regeneration in situ, suggesting its used as a promising substitute in bone tissue engineering.

Silk fibroin magnetoactive nanocomposite films and membranes for dynamic bone tissue engineering strategies

Bone-related diseases are one of the most common health conditions that limit the quality of life of elderly. Novel materials for bone tissue engineering that actively assist on the regeneration of bone tissue are thus needed. In this work, magnetoactive scaffolds comprised of silk fibroin (SF) with different content of filler – cobalt ferrite nanoparticles – were produced by solvent-casting and electrospinning technique and further evaluated for bone cells growth under static and dynamic magnetic stimulation. The materials were evaluated for their enhanced electric properties, analyzed through the silk fibroin β-sheet content, an important factor for the envisaged cell stimulation strategy, which are in much higher content in films (~50%) than in electrospun fibers (~25%). Cell culture experiments under varying magnetic field, induced a magneto-mechanical stimulation on the materials, hence on cells, promoting improved cell viability after 4 days of culture. The scaffold morphology was found to play an important role in pre-osteoblast proliferation rate, being larger for cells growing on films, which is related to the topography of the material but also to the increased β-sheet content. It is shown that the use of magnetic cues on magnetoactive biocompatible scaffolds is a promising strategy for remote stimulation of bone for its regeneration. Statement of Significance The use of physical stimuli such as electrical and mechanical cues have been proven a powerful tool for bone tissue engineering applications, but the use of magnetic cues on magnetoactive scaffolds has been scarcely reported. This is considered a promising strategy for remote stimulation of bone for its regeneration. The present study provides data on the evaluation of biocompatible silk fibroin scaffolds for bone cells’ proper adhesion and proliferation upon the application of a magnetic stimuli using a custom-made magnetic bioreactor. The conditions created promote a biomimetic microenvironment through the application of a mechanical and electrical cues to cells. This approach is interesting since while scaffolds mimics the morphology of bone, the magnetic bioreactor provides the physical environment of bone, i.e. piezoelectricity, by generating electrical and mechanical cues with magnetic stimulation.

Adhesion, intracellular signalling and osteogenic differentiation of mesenchymal progenitor cells and preosteoblasts on poly(epsilon)caprolactone films functionalized by peptides derived from fibronectin and/or BMP-9.

Biomaterials that can control the behaviour of stem cells play a major role in regenerative medicine and tissue engineering. We previously showed that poly(epsilon)caprolactone (PCL) films functionalized with adhesive peptides containing sequences of both cell binding domain (RGD) and synergistic site (PHSRN) of the fibronectin (pFibro) enhanced the osteoblastic commitment of C3H10T1/2 mesenchymal progenitor cells (C3H10T1/2 cells) induced by soluble BMP-9 or its derived peptide SpBMP-9. Here, the effect of PCL films functionalized with pFibro and/or SpBMP-9 or its negative peptide NSpBMP-9 on adhesion and intracellular signalling of C3H10T1/2 cells was determined. The differentiation of adherent C3H10T1/2 cells and MC3T3-E1 preosteoblasts into osteoblasts was also analysed with or without IGF-2, since this growth factor can favour the osteoblastic differentiation induced by BMP-9. We found that pFibro and SpBMP-9 co-functionalization on PCL films promoted the adhesion of C3H10T1/2 cells with well-organized focal adhesion points and FAK activation. In these mesenchymal progenitor cells, PCL-SpBMP-9 and PCL-pFibro/SpBMP-9 induced the activation and nuclear translocation of Smad 1/5 after 4h, and enhanced the protein expression of RUNX2 (3 d) and alkaline phosphatase (ALP) activity (7 d), two markers of osteoblast differentiation. No PPARγ, a marker of adipogenic differentiation, was detected. C3H10T1/2 cells attached to PCL-SpBMP-9 also contained more SOX9, a marker of chondroblastic lineage, compared with other experimental conditions. The use of inactive peptides NSpBMP-9 confirmed the specificity and effectiveness of SpBMP-9 on cell adhesion, intracellular signalling and osteoblastic differentiation. Adding IGF-2 only significantly improved the differentiation of MC3T3-E1 preosteoblasts into osteoblasts as shown by the increase in gene expression encoding Osterix (mRNA Sp7) and ALP (mRNA Alpl), probably because of the lack of serum in the medium. Therefore, material surface co-functionalized with pFibro and SpBMP-9 could be most useful for developing scaffolds with both osseointegrative and osteoinductive properties for bone application and tissue engineering strategy when combined with IGF-2 in serum free medium.

Degradable Poly(Methyl Methacrylate)-co-Methacrylic Acid Nanoparticles for Controlled Delivery of Growth Factors for Bone Regeneration.

Bone tissue engineering strategies have been developed to address the limitations of the current gold standard treatment options for bone-related disorders. These systems consist of an engineered scaffold that mimics the extracellular matrix and provides an architecture to guide the natural bone regeneration process, and incorporated growth factors that enhance cell recruitment and ingress into the scaffold and promote the osteogenic differentiation of stem cells and angiogenesis. In particular, the osteogenic growth factor bone morphogenetic protein 2 (BMP-2) has been widely studied as a potent agent to improve bone regeneration. A key challenge in growth factor delivery is that the growth factors must reach their target sites without losing bioactivity and remain in the location for an extended period to effectively aid in the formation of new bone. Protein incorporation into nanoparticles can both protect protein bioactivity and enable its sustained release. In this study, a poly(methyl methacrylate-co-methacrylic acid) nanoparticle-based system was synthesized incorporating a custom poly(ethylene glycol) dimethacrylate crosslinker. It was demonstrated that the nanoparticle degradation rate can be controlled by tuning the number of hydrolytically degradable ester units along the crosslinker. We also showed that the nanoparticles had high affinity for a model protein for BMP-2, and optimal conditions for maximum protein loading efficiency were elucidated. Ultimately, the proposed system and its high degree of tunability can be applied to a wide range of growth factors and tissue engineering applications. Impact Statement In this study, we developed a novel method of synthesizing nanoparticles with tunable degradation rates through the incorporation of a custom synthesized, hydrolytically degradable crosslinker. In addition, we demonstrated the affinity of the synthesized nanoparticles for a model protein for bone morphogenetic protein 2 (BMP-2). The tunability of these nanoparticles can be used to develop complex tissue engineering systems, for example, for the delivery of multiple growth factors involved at different stages of the bone regeneration process.

Scaffolds and coatings for bone regeneration.

Bone tissue has an astonishing self-healing capacity yet only for non-critical size defects (<6 mm) and clinical intervention is needed for critical-size defects and beyond that along with non-union bone fractures and bone defects larger than critical size represent a major healthcare problem. Autografts are, still, being used as preferred to treat large bone defects. Mostly, due to the presence of living differentiated and progenitor cells, its osteogenic, osteoinductive and osteoconductive properties that allow osteogenesis, vascularization, and provide structural support. Bone tissue engineering strategies have been proposed to overcome the limited supply of grafts. Complete and successful bone regeneration can be influenced by several factors namely: the age of the patient, health, gender and is expected that the ideal scaffold for bone regeneration combines factors such as bioactivity and osteoinductivity. The commercially available products have as their main function the replacement of bone. Moreover, scaffolds still present limitations including poor osteointegration and limited vascularization. The introduction of pores in scaffolds are being used to promote the osteointegration as it allows cell and vessel infiltration. Moreover, combinations with growth factors or coatings have been explored as they can improve the osteoconductive and osteoinductive properties of the scaffold. This review focuses on the bone defects treatments and on the research of scaffolds for bone regeneration. Moreover, it summarizes the latest progress in the development of coatings used in bone tissue engineering. Despite the interesting advances which include the development of hybrid scaffolds, there are still important challenges that need to be addressed in order to fasten translation of scaffolds into the clinical scenario. Finally, we must reflect on the main challenges for bone tissue regeneration. There is a need to achieve a proper mechanical properties to bear the load of movements; have a scaffolds with a structure that fit the bone anatomy.

Fat Graft Enrichment Strategies: A Systematic Review.

Autologous fat grafting is a dynamic modality used in plastic surgery as an adjunct to improve functional and aesthetic form. However, current practices in fat grafting for soft-tissue augmentation are plagued by tremendous variability in long-term graft retention, resulting in suboptimal outcomes and repetitive procedures. This systematic review identifies and critically appraises the evidence for various enrichment strategies that can be used to augment and improve the viability of fat grafts. A comprehensive literature search of the Medline and PubMed databases was conducted for animal and human studies published through October of 2017 with multiple search terms related to adipose graft enrichment agents encompassing growth factors, platelet-rich plasma, adipose-derived and bone marrow stem cells, gene therapy, tissue engineering, and other strategies. Data on level of evidence, techniques, complications, and outcomes were collected. A total of 1382 articles were identified, of which 147 met inclusion criteria. The majority of enrichment strategies demonstrated positive benefit for fat graft survival, particularly with growth factors and adipose-derived stem cell enrichment. Platelet-rich plasma and adipose-derived stem cells had the strongest evidence to support efficacy in human studies and may demonstrate a dose-dependent effect. Improved understanding of enrichment strategies contributing to fat graft survival can help to optimize safety and outcomes. Controlled clinical studies are lacking, and future studies should examine factors influencing graft survival through controlled clinical trials in order to establish safety and to obtain consistent outcomes.

Recent advances toward understanding the role of transplanted stem cells in tissue-engineered regeneration of musculoskeletal tissues.

Stem cell–based tissue engineering is poised to revolutionize the treatment of musculoskeletal injuries. However, in order to overcome scientific, practical, and regulatory obstacles and optimize therapeutic strategies, it is essential to better understand the mechanisms underlying the pro-regenerative effects of stem cells. There has been an attempted paradigm shift within the last decade to think of transplanted stem cells as “medicinal” therapies that orchestrate healing on the basis of their secretome and immunomodulatory profiles rather than acting as bona fide stem cells that proliferate, differentiate, and directly produce matrix to form de novo tissues. Yet the majority of current bone and skeletal muscle tissue engineering strategies are still premised on a direct contribution of stem cells as building blocks to tissue regeneration. Our review of the recent literature finds that researchers continue to focus on the quantification of de novo bone/skeletal muscle tissue following treatment and few studies aim to address this mechanistic conundrum directly. The dichotomy of thought is reflected in the diversity of new advances ranging from in situ three-dimensional bioprinting to a focus on exosomes and extracellular vesicles. However, recent findings elucidating the role of the immune system in tissue regeneration combined with novel imaging platform technologies will have a profound impact on our future understanding of how stem cells promote healing following biomaterial-mediated delivery to defect sites.

3D printed Sr-containing composite scaffolds: Effect of structural design and material formulation towards new strategies for bone tissue engineering

The use of composite materials, processed as 3D tissue-like scaffolds, has been widely investigated as a promising strategy for bone tissue engineering applications. Also, additive manufacturing technologies such as fused deposition modelling (FDM) have greatly contributed to the manufacture of patient-specific scaffolds with predefined pore structures and intricate geometries. However, conventional FDM techniques require the use of materials exclusively in the form of filaments, which in order to produce composite scaffolds lead to additional costs for the fabrication of precursor filaments as well as multi-step production methods. In this study, we propose the use of an advantageous extrusion-based printing technology, which provides the opportunity to easily co-print biomaterials, starting from their raw forms, and by using a single-step manufacturing and solvent-free process. Poly(e-caprolactone) (PCL), an FDA approved biodegradable material, was used as polymeric matrix while hydroxyapatite (HA) and strontium substituted HA (SrHA), at various contents were introduced as a bioactive reinforcing phase capable of mimicking the mineral phase of natural bone. Three different architectures for each material formulation were designed, and subsequently the effect of composition variations and structural designs was analysed in terms of physico-chemical, mechanical and biological performance. A correlation between architecture and compressive modulus, regardless the formulation tested, was observed demonstrating how the laydown pattern influences the resulting 3D printed scaffolds’ stiffness. Furthermore, in vitro cell culture by using TERT Human Mesenchymal Stromal Cells (hTERT-MSCs) revealed that Sr-containing composite scaffolds showed greater levels of mineralisation and osteogenic potential in comparison to bare PCL and pure HA.

Immobilization of nanocarriers within a porous chitosan scaffold for the sustained delivery of growth factors in bone tissue engineering applications

To guide the natural bone regeneration process, bone tissue engineering strategies rely on the development of a scaffold architecture that mimics the extracellular matrix and incorporates important extracellular signaling molecules, which promote fracture healing and bone formation pathways. Incorporation of growth factors into particles embedded within the scaffold can offer both protection of protein bioactivity and a sustained release profile. In this work, a novel method to immobilize carrier nanoparticles within scaffold pores is proposed. A biodegradable, osteoconductive, porous chitosan scaffold was fabricated via the "freeze-drying method," leading to scaffolds with a storage modulus of 8.5 kPa and 300 μm pores, in line with existing bone scaffold properties. Next, poly(methyl methacrylate-co-methacrylic acid) nanoparticles were synthesized and immobilized to the scaffold via carbodiimide-crosslinker chemistry. A fluorescent imaging study confirmed that the conventional methods of protein and nanocarrier incorporation into scaffolds can lead to over 60% diffusion out of the scaffold within the first 5 min of implantation, and total disappearance within 4 weeks. The novel method of nanocarrier immobilization to the scaffold backbone via carbodiimide-crosslinker chemistry allows full retention of particles for up to 4 weeks within the scaffold bulk, with no negative effects on the viability and proliferation of human umbilical vein endothelial cells.

Bone Tissue Regeneration in the Oral and Maxillofacial Region: A Review on the Application of Stem Cells and New Strategies to Improve Vascularization.

Bone tissue engineering techniques are a promising alternative for the use of autologous bone grafts to reconstruct bone defects in the oral and maxillofacial region. However, for successful bone regeneration, adequate vascularization is a prerequisite. This review presents and discusses the application of stem cells and new strategies to improve vascularization, which may lead to feasible clinical applications. Multiple sources of stem cells have been investigated for bone tissue engineering. The stromal vascular fraction (SVF) of human adipose tissue is considered a promising single source for a heterogeneous population of essential cells with, amongst others, osteogenic and angiogenic potential. Enhanced vascularization of tissue-engineered grafts can be achieved by different mechanisms: vascular ingrowth directed from the surrounding host tissue to the implanted graft, vice versa, or concomitantly. Vascular ingrowth into the implanted graft can be enhanced by (i) optimizing the material properties of scaffolds and (ii) their bioactivation by incorporation of growth factors or cell seeding. Vascular ingrowth directed from the implanted graft towards the host tissue can be achieved by incorporating the graft with either (i) preformed microvascular networks or (ii) microvascular fragments (MF). The latter may have stimulating actions on both vascular ingrowth and outgrowth, since they contain angiogenic stem cells like SVF, as well as vascularized matrix fragments. Both adipose tissue-derived SVF and MF are cell sources with clinical feasibility due to their large quantities that can be harvested and applied in a one-step surgical procedure. During the past years, important advancements of stem cell application and vascularization in bone tissue regeneration have been made. The development of engineered in vitro 3D models mimicking the bone defect environment would facilitate new strategies in bone tissue engineering. Successful clinical application requires innovative future investigations enhancing vascularization.

Gelatin-Based Microribbon Hydrogels Guided Mesenchymal Stem Cells to Undergo Endochondral Ossification In Vivo with Bone-Mimicking Mechanical Strength

Most stem cell–based bone tissue engineering strategies to date yield bone through direct bone formation, which mimics intramembranous ossification. However, bone injuries often affect long bones which are formed through endochondral ossification, involving an initial cartilage template formation followed by remodeling to form bones. There remains a critical need to develop scaffolds that enhance stem cell–based bone formation through endochondral ossification with bone-mimicking mechanical strength. Here we evaluated the potential of gelatin-based microribbons (μRBs) as macroporous scaffolds for enhancing human mesenchymal stem cell (MSC)–based bone formation through endochondral ossification. This material platform was compared with conventional gelatin hydrogels (HGs) as controls. MSCs were encapsulated in μRB or HG scaffolds, primed in chondrogenic medium in vitro for 2 weeks, and then implanted in a mouse subcutaneous model with no additional factors. μRB scaffolds supported fast cartilage deposition by MSCs, which was completely remodeled and replaced by mineralized bone. Impressively, the compressive moduli of MSC-seeded μRB scaffolds increased from 10 to 3224 kPa by week 11, a range that mimics native bone. In comparison, while HG supported endochondral ossification, the speed was much slower, with less matrix deposition and only a modest increase in compressive modulus to 269 kPa. These results validate gelatin μRBs as a promising scaffold for repairing long bone defects by guiding robust endochondral ossification. Natural bone development and healing occurs through two distinct pathways: intramembranous ossification and endochondral ossification. Most bone injuries affect long bones, which are formed through endochondral ossification, involving an initial cartilage template formation followed by remodeling to form bones. However, scaffolds that can guide stem cell–based bone formation through endochondral ossification with bone-mimicking mechanical strength remain lacking. Here we report that macroporous gelatin-based microribbons (μRBs) accelerate endochondral ossification by human mesenchymal stem cells (MSCs) in vivo using a mouse subcutaneous model. Impressively, the mechanical properties of MSC-seeded μRB scaffolds increased over 300-fold over 11 weeks to bone-mimicking range, whereas conventional gelatin hydrogel controls reached less than 10% of the bone modulus. These results validate gelatin μRBs as a promising novel scaffold for repairing long bone defects by guiding robust endochondral ossification.

Dipyridamole-loaded 3D-printed bioceramic scaffolds stimulate pediatric bone regeneration in vivo without disruption of craniofacial growth through facial maturity

This study investigates a comprehensive model of bone regeneration capacity of dypiridamole-loaded 3D-printed bioceramic (DIPY-3DPBC) scaffolds composed of 100% beta-tricalcium phosphate (β –TCP) in an immature rabbit model through the time of facial maturity. The efficacy of this construct was compared to autologous bone graft, the clinical standard of care in pediatric craniofacial reconstruction, with attention paid to volume of regenerated bone by 3D reconstruction, histologic and mechanical properties of regenerated bone, and long-term safety regarding potential craniofacial growth restriction. Additionally, long-term degradation of scaffold constructs was evaluated. At 24 weeks in vivo, DIPY-3DPBC scaffolds demonstrated volumetrically significant osteogenic regeneration of calvarial and alveolar defects comparable to autogenous bone graft with favorable biodegradation of the bioactive ceramic component in vivo. Characterization of regenerated bone reveals osteogenesis of organized, vascularized bone with histologic and mechanical characteristics comparable to native bone. Radiographic and histologic analyses were consistent with patent craniofacial sutures. Lastly, through application of 3D morphometric facial surface analysis, our results support that DIPY-3DPBC scaffolds do not cause premature closure of sutures and preserve normal craniofacial growth. Based on this novel evaluation model, this DIPY-3DPBC scaffold strategy is a promising candidate as a safe, efficacious pediatric bone tissue engineering strategy.

Mesenchymal Stromal Cell-Based Bone Regeneration Therapies: From Cell Transplantation and Tissue Engineering to Therapeutic Secretomes and Extracellular Vesicles.

Effective regeneration of bone defects often presents significant challenges, particularly in patients with decreased tissue regeneration capacity due to extensive trauma, disease, and/or advanced age. A number of studies have focused on enhancing bone regeneration by applying mesenchymal stromal cells (MSCs) or MSC-based bone tissue engineering strategies. However, translation of these approaches from basic research findings to clinical use has been hampered by the limited understanding of MSC therapeutic actions and complexities, as well as costs related to the manufacturing, regulatory approval, and clinical use of living cells and engineered tissues. More recently, a shift from the view of MSCs directly contributing to tissue regeneration toward appreciating MSCs as “cell factories” that secrete a variety of bioactive molecules and extracellular vesicles with trophic and immunomodulatory activities has steered research into new MSC-based, “cell-free” therapeutic modalities. The current review recapitulates recent developments, challenges, and future perspectives of these various MSC-based bone tissue engineering and regeneration strategies.

Reconstruction of segmental mandibular defects: Current procedures and perspectives.

BackgroundThe reconstruction of segmental mandibular defects remains a challenge for the reconstructive surgeon, from both a functional and an esthetic point of view.MethodsThis clinical review examines the different techniques currently in use for mandibular reconstruction as related to a range of etiologies, including the different bone donor sites, the alternatives to free flaps (FFs), as well as the contribution of computer‐assisted surgery. Recent progress and the perspectives in bone tissue engineering (BTE) are also discussed.ResultsOsseous FF allows reliable and satisfying outcomes. However, locoregional flap, distraction osteogenesis, or even induced membrane techniques are other potential options in less favorable cases. Obtaining an engineered bone with satisfactory mechanical properties and sufficient vascular supply requires further investigations.ConclusionsOsseous FF procedure remains the gold standard for segmental mandible reconstruction. BTE strategies offer promising alternatives.

Stimulation of Osteogenic Differentiation of Induced Pluripotent Stem Cells (iPSCs) Using Bioactive Glasses: An in vitro Study.

Selection and use of an optimal cell source for bone tissue engineering (BTE) remain a challenging issue; the invention of induced pluripotent stem cells (iPSCs) have created new hopes on this regard. At the present study, we attempted to show the usability of iPSCs in combination with bioactive glasses (BGs) for bone regeneration applications. For this aim, iPSCs were cultured and incubated with the strontium and cobalt-containing BGs for different intervals (1, 5, and 7 days). The cell cytotoxicity and attachment were assessed using MTT assay and scanning electron microscopy (SEM), respectively. Moreover, the osteogenic differentiation of iPSCs seeded onto the glasses was evaluated using alkaline phosphatase (ALP) activity assay and real-time PCR. The obtained results clarified that although the cell viability is decreased during a 7 day period, the iPSCs could adhere and expand onto the BGs particles and over-express the osteogenic markers, including osteocalcin, osteonectin, and Runx2. Based on the data, we conclude that iPSCs in a combination of BGs can be considered as a potential candidate for BTE strategies.

Applications of X-ray computed tomography for the evaluation of biomaterial-mediated bone regeneration in critical-sized defects.

Bone as such displays an intrinsic regenerative potential following fracture; however, this capacity is limited with large bone defects that cannot heal spontaneously. The management of critical-sized bone defects remains a major clinical and socioeconomic need with osteoregenerative biomaterials constantly under development aiming at promoting and enhancing bone healing. X-ray computed tomography (XCT) has become a standard and essential tool for quantifying structure-function relationships in bone and biomaterials, facilitating the development of novel bone tissue engineering strategies. This paper presents recent advancements in XCT analysis of biomaterial-mediated bone regeneration. As a noninvasive and nondestructive technique, XCT allows for qualitative and quantitative evaluation of three-dimensional (3D) scaffolds and biomaterial microarchitecture, bone growth into the scaffold as well as the 3D characterisation of biomaterial degradation and bone regeneration in vitro and in vivo. Furthermore, in combination with in situ mechanical testing and digital volume correlation (DVC), XCT demonstrated its potential to better understand the bone-biomaterial interactions and local mechanics of bone regeneration during the healing process in relation to the regeneration achieved in vivo, which will likely provide valuable knowledge for the development and optimisation of novel osteoregenerative biomaterials. LAY DESCRIPTION: Bone, being a dynamically adaptable material, displays excellent regenerative properties following fracture. However, the self-healing capacity of bone becomes more difficult with large bone defects. Those defects are common and occur in many clinical situations; hence, biomaterials are mostly used to restore both bone structure and function in the defect site. X-ray computed tomography (XCT) is a powerful tool to evaluate bone regeneration in critical-sized defects after the implantation of biomaterials, allowing to an improved understanding of the regeneration process following different bone tissue engineering approaches. This paper focuses on recent advancements in XCT analysis to characterise biomaterial-mediated bone regeneration in critical-sized defects. XCT supports three-dimensional (3D) analysis of biomaterials, scaffolds and regenerated bone microarchitecture, as well as bone ingrowth into the scaffold. As a nondestructive technique, XCT allows for a 3D characterisation of biomaterial degradation and bone regeneration over time. In addition, XCT combined with in situ mechanical experiments and digital volume correlation (DVC) provides a 3D evaluation and quantification of bone-biomaterial interactions and deformation mechanisms during the regeneration process. This remains essential for the development and enhancement of novel biomaterials able to produce bone that is comparable with the native tissue they aim to replace.

Bioengineering strategies for bone and cartilage tissue regeneration using growth factors and stem cells.

Bone and cartilage tissue engineering is an integrative approach that is inspired by the phenomena associated with wound healing. In this respect, growth factors have emerged as important moieties for the control and regulation of this process. Growth factors act as mediators and control the important physiological functions of bone regeneration. Herein, we discuss the importance of growth factors in bone and cartilage tissue engineering, their loading and delivery strategies, release kinetics, and their integration with biomaterials and stem cells to heal bone fractures. We also highlighted the role of growth factors in the determination of the bone tissue microenvironment based on the reciprocal signaling with cells and biomaterial scaffolds on which future bone and cartilage tissue engineering technologies and medical devices will be based upon.

Fabrication and In Vitro Evaluation of 3D Printed Porous Polyetherimide Scaffolds for Bone Tissue Engineering.

For bone tissue engineering, the porous scaffold should provide a biocompatible environment for cell adhesion, proliferation, and differentiation and match the mechanical properties of native bone tissue. In this work, we fabricated porous polyetherimide (PEI) scaffolds using a three-dimensional (3D) printing system, and the pore size was set as 800 μm. The morphology of 3D PEI scaffolds was characterized by the scanning electron microscope. To investigate the mechanical properties of the 3D PEI scaffold, the compressive mechanical test was performed via an electronic universal testing system. For the in vitro cell experiment, bone marrow stromal cells (BMSCs) were cultured on the surface of the 3D PEI scaffold and PEI slice, and cytotoxicity, cell adhesion, and cell proliferation were detected to verify their biocompatibility. Besides, the alkaline phosphatase staining and Alizarin Red staining were performed on the BMSCs of different samples to evaluate the osteogenic differentiation. Through these studies, we found that the 3D PEI scaffold showed an interconnected porous structure, which was consistent with the design. The elastic modulus of the 3D PEI scaffold (941.33 ± 65.26 MPa) falls in the range of modulus for the native cancellous bone. Moreover, the cell proliferation and morphology on the 3D PEI scaffold were better than those on the PEI slice, which revealed that the porous scaffold has good biocompatibility and that no toxic substances were produced during the progress of high-temperature 3D printing. The osteogenic differentiation level of the 3D PEI scaffold and PEI slice was equal and ordinary. All of these results suggest the 3D printed PEI scaffold would be a potential strategy for bone tissue engineering.

Is There a Noninvasive Source of MSCs Isolated with GMP Methods with Better Osteogenic Potential?

Background A new trend in the treatment for alveolar clefts in patients with cleft lip and palate involves the use of bone tissue engineering strategies to reduce or eliminate the morbidity associated with autologous bone grafting. The use of mesenchymal stem cells—autologous cells obtained from tissues such as bone marrow and fat—combined with various biomaterials has been proposed as a viable option for use in cleft patients. However, invasive procedures are necessary to obtain the mesenchymal stem cells from these two sources. To eliminate donor site morbidity, noninvasive stem cell sources such as the umbilical cord, orbicularis oris muscle, and deciduous dental pulp have been studied for use in alveolar cleft bone tissue engineering. In this study, we evaluate the osteogenic potential of these various stem cell types. Methods Ten cellular strains obtained from each different source (umbilical cord, orbicularis oris muscle, or deciduous dental pulp) were induced to osteogenic differentiation in vitro, and the bone matrix deposition of each primary culture was quantified. To evaluate whether greater osteogenic potential of the established mesenchymal stem cell strains was associated with an increase in the expression profile of neural crest genes, real-time qPCR was performed on the following genes: SRY-box 9, SRY-box 10, nerve growth factor receptor, transcription factor AP-2 alpha, and paired box 3. Results The mesenchymal stem cells obtained from deciduous dental pulp and orbicularis oris muscle demonstrated increased osteogenic potential with significantly more extracellular bone matrix deposition when compared to primary cultures obtained from the umbilical cord after twenty-one days in culture (p = 0.007 and p = 0.005, respectively). The paired box 3 gene was more highly expressed in the MSCs obtained from deciduous dental pulp and orbicularis oris muscle than in those obtained from the umbilical cord. Conclusion These results suggest that deciduous dental pulp and orbicularis oris muscle stem cells demonstrate superior osteogenic differentiation potential relative to umbilical cord-derived stem cells and that this increased potential is related to their neural crest origins. Based on these observations, and the distinct translational advantage of incorporating stem cells from noninvasive tissue sources into tissue engineering protocols, greater study of these specific cell lines in the setting of alveolar cleft repair is indicated.