Osteoinductive calcium phosphate with submicron topography as bone graft substitute for maxillary sinus floor augmentation: A translational study.

OBJECTIVES: The aim of this study was the preclinical and clinical evaluation of osteoinductive calcium phosphate with submicron surface topography as a bone graft substitute for maxillary sinus floor augmentation (MSFA). MATERIAL AND METHODS: A preclinical sheep model of MSFA was used to compare a calcium phosphate with submicron needle-shaped topography (BCPN , MagnetOs Granules, Kuros Biosciences BV) to a calcium phosphate with submicron grain-shaped topography (BCPG ) and autologous bone graft (ABG) as controls. Secondly, a 10-patient, prospective, randomized, controlled trial was performed to compare BCPN to ABG in MSFA with two-stage implant placement. RESULTS: The pre-clinical study demonstrated that both BCPN and BCPG were highly biocompatible, supported bony ingrowth with direct bone apposition against the material, and exhibited bone formation as early as 3 weeks post-implantation. However, BCPN demonstrated significantly more bone formation than BCPG at the study endpoint of 12 weeks. Only BCPN reached an equivalent amount of bone formation in the available space and a greater proportion of calcified material (bone + graft material) in the maxillary sinus compared to the "gold standard" ABG after 12 weeks. These results were validated in a small prospective clinical study, in which BCPN was found comparable to ABG in implant stability, bone height, new bone formation in trephine core biopsies, and overall clinical outcome. CONCLUSION: This translational work demonstrates that osteoinductive calcium phosphates are promising bone graft substitutes for MSFA, whereas their bone-forming potential depends on the design of their surface features. Netherlands Trial Register, NL6436.

Mapping the microcarrier design pathway to modernise clinical mesenchymal stromal cell expansion.

Microcarrier expansion systems show exciting potential to revolutionise mesenchymal stromal cell (MSC)-based clinical therapies by providing an opportunity for economical large-scale expansion of donor- and patient-derived cells. The poor reproducibility and efficiency of cell expansion on commercial polystyrene microcarriers have driven the development of novel microcarriers with tuneable physical, mechanical, and cell-instructive properties. These new microcarriers show innovation toward improving cell expansion outcomes, although their limited biological characterisation and compatibility with dynamic culture systems suggest the need to realign the microcarrier design pathway. Clear headway has been made toward developing infrastructure necessary for scaling up these technologies; however, key challenges remain in characterising the wholistic effects of microcarrier properties on the biological fate and function of expanded MSCs.

Clinical Applicability of Visible Light-Mediated Cross-linking for Structural Soft Tissue Reconstruction.

Visible light-mediated cross-linking has utility for enhancing the structural capacity and shape fidelity of laboratory-based polymers. With increased light penetration and cross-linking speed, there is opportunity to extend future applications into clinical spheres. This study evaluated the utility of a ruthenium/sodium persulfate photocross-linking system for increasing structural control in heterogeneous living tissues as an example, focusing on unmodified patient-derived lipoaspirate for soft tissue reconstruction. Freshly-isolated tissue is photocross-linked, then the molar abundance of dityrosine bonds is measured using liquid chromatography tandem mass spectrometry and the resulting structural integrity assessed. The cell function and tissue survival of photocross-linked grafts is evaluated ex vivo and in vivo, with tissue integration and vascularization assessed using histology and microcomputed tomography. The photocross-linking strategy is tailorable, allowing progressive increases in the structural fidelity of lipoaspirate, as measured by a stepwise reduction in fiber diameter, increased graft porosity and reduced variation in graft resorption. There is an increase in dityrosine bond formation with increasing photoinitiator concentration, and tissue homeostasis is achieved ex vivo, with vascular cell infiltration and vessel formation in vivo. These data demonstrate the capability and applicability of photocrosslinking strategies for improving structural control in clinically-relevant settings, potentially achieving more desirable patient outcomes using minimal manipulation in surgical procedures.

Bone Regeneration in a Large Animal Model Featuring a Modular Off-the-Shelf Soft Callus Mimetic.

Implantation of engineered cartilage with soft callus features triggers remodeling to bone tissue via endochondral bone regeneration (EBR). Thus far, EBR has not progressed to the level of large animals on the axis of clinical translation. Herein, the feasibility of EBR is aimed for a critical-sized defect in a large animal model. Chondrogenesis is first induced in goat-derived multipotent mesenchymal stromal cells (MSCs) by fine-tuning the cellular differentiation process. Through a unique devitalization process, two off-the-shelf constructs aimed to recapitulate the different stages of the transient cartilaginous soft callus template in EBR are generated. To evaluate bone regeneration, the materials are implanted in an adapted bilateral iliac crest defect model in goats, featuring a novel titanium star-shaped spacer. After 3 months, the group at the more advanced differentiation stage shows remarkable regenerative capacity, with comparable amounts of bone regeneration as the autograft group. In contrast, while the biomaterial mimicking the earlier stages of chondrogenesis shows improved regeneration compared to the negative controls, this is subpar compared to the more advanced material. Concluding, EBR is attainable in large animals with a soft callus mimetic material that leads to fast conversion into centimeter-scale bone, which prospects successful implementation in the human clinics.

Pristine gelatin incorporation as a strategy to enhance the biofunctionality of poly(vinyl alcohol)-based hydrogels for tissue engineering applications.

Synthetic polymers, such as poly(vinyl alcohol) (PVA), are popular biomaterials for the fabrication of hydrogels for tissue engineering and regenerative medicine (TERM) applications, as they provide excellent control over the physico-chemical properties of the hydrogel. However, their bioinert nature is known to limit cell-biomaterial interactions by hindering cell infiltration, blood vessel recruitment and potentially limiting their integration with the host tissue. Efforts in the field have therefore focused on increasing the biofunctionality of synthetic hydrogels, without limiting the advantages associated with their tailorability and controlled release capacity. The aim of this study was to investigate the suitability of pristine gelatin to enhance the biofunctionality of tyraminated PVA (PVA-Tyr) hydrogels, by promoting cell infiltration and host blood vessel recruitment for TERM applications. Pure PVA-Tyr hydrogels and PVA-Tyr hydrogels incorporated with vascular endothelial growth factor (VEGF), a well-known pro-angiogenic stimulus, were used for comparison. Incorporating increasing concentrations of VEGF (0.01-10 µg mL-1) or gelatin (0.01-5 wt%) did not influence the physical properties of PVA-Tyr hydrogels. However, their presence within the polymer network (>0.1 µg mL-1 VEGF and >0.1 wt% gelatin) promoted endothelial cell interactions with the hydrogels. The covalent binding of unmodified gelatin or VEGF to the PVA-Tyr network did not hamper their inherent bioactivity, as they both promoted angiogenesis in a chick chorioallantoic membrane (CAM) assay, performing comparably with the unbound VEGF control. When the PVA-Tyr hydrogels were implanted subcutaneously in mice, it was observed that cell infiltration into the hydrogels was possible in the absence of gelatin or VEGF at 1- or 3-weeks post-implantation, highlighting a clear difference between in vitro an in vivo cell-biomaterial interaction. Nevertheless, the presence of gelatin or VEGF was necessary to enhance blood vessel recruitment and infiltration, although no significant difference was observed between these two biological molecules. Overall, this study highlights the potential of gelatin as a standalone pro-angiogenic cue to enhance biofunctionality of synthetic hydrogels and provides promise for their use in a variety of TERM applications.

Harnessing Macromolecular Chemistry to Design Hydrogel Micro- and Macro-Environments.

Cell encapsulation within three-dimensional hydrogels is a promising approach to mimic tissues. However, true biomimicry of the intricate microenvironment, biophysical and biochemical gradients, and the macroscale hierarchical spatial organizations of native tissues is an unmet challenge within tissue engineering. This review provides an overview of the macromolecular chemistries that have been applied toward the design of cell-friendly hydrogels, as well as their application toward controlling biophysical and biochemical bulk and gradient properties of the microenvironment. Furthermore, biofabrication technologies provide the opportunity to simultaneously replicate macroscale features of native tissues. Biofabrication strategies are reviewed in detail with a particular focus on the compatibility of these strategies with the current macromolecular toolkit described for hydrogel design and the challenges associated with their clinical translation. This review identifies that the convergence of the ever-expanding macromolecular toolkit and technological advancements within the field of biofabrication, along with an improved biological understanding, represents a promising strategy toward the successful tissue regeneration.

Spatial-Selective Volumetric 4D Printing and Single-Photon Grafting of Biomolecules within Centimeter-Scale Hydrogels via Tomographic Manufacturing.

Conventional additive manufacturing and biofabrication techniques are unable to edit the chemicophysical properties of the printed object postprinting. Herein, a new approach is presented, leveraging light-based volumetric printing as a tool to spatially pattern any biomolecule of interest in custom-designed geometries even across large, centimeter-scale hydrogels. As biomaterial platform, a gelatin norbornene resin is developed with tunable mechanical properties suitable for tissue engineering applications. The resin can be volumetrically printed within seconds at high resolution (23.68 ± 10.75 µm). Thiol-ene click chemistry allows on-demand photografting of thiolated compounds postprinting, from small to large (bio)molecules (e.g., fluorescent dyes or growth factors). These molecules are covalently attached into printed structures using volumetric light projections, forming 3D geometries with high spatiotemporal control and ≈50 µm resolution. As a proof of concept, vascular endothelial growth factor is locally photografted into a bioprinted construct and demonstrated region-dependent enhanced adhesion and network formation of endothelial cells. This technology paves the way toward the precise spatiotemporal biofunctionalization and modification of the chemical composition of (bio)printed constructs to better guide cell behavior, build bioactive cue gradients. Moreover, it opens future possibilities for 4D printing to mimic the dynamic changes in morphogen presentation natively experienced in biological tissues.

Acceleration of Bone Regeneration Induced by a Soft-Callus Mimetic Material.

Clinical implementation of endochondral bone regeneration (EBR) would benefit from the engineering of devitalized cartilaginous constructs of allogeneic origins. Nevertheless, development of effective devitalization strategies that preserves extracellular matrix (ECM) is still challenging. The aim of this study is to investigate EBR induced by devitalized, soft callus-mimetic spheroids. To challenge the translatability of this approach, the constructs are generated using an allogeneic cell source. Neo-bone formation is evaluated in an immunocompetent rat model, subcutaneously and in a critical size femur defect. Living spheroids are used as controls. Also, the effect of spheroid maturation towards hypertrophy is evaluated. The devitalization procedure successfully induces cell death without affecting ECM composition or bioactivity. In vivo, a larger amount of neo-bone formation is observed for the devitalized chondrogenic group both ectopically and orthotopically. In the femur defect, accelerated bone regeneration is observed in the devitalized chondrogenic group, where defect bridging is observed 4 weeks post-implantation. The authors' results show, for the first time, a dramatic increase in the rate of bone formation induced by devitalized soft callus-mimetics. These findings pave the way for the development of a new generation of allogeneic, "off-the-shelf" products for EBR, which are suitable for the treatment of every patient.

Development and Characterization of Gelatin-Norbornene Bioink to Understand the Interplay between Physical Architecture and Micro-Capillary Formation in Biofabricated Vascularized Constructs.

The principle challenge for engineering viable, cell-laden hydrogel constructs of clinically-relevant size, is rapid vascularization, in order to moderate the finite capacity of passive nutrient diffusion. A multiscale vascular approach, with large open channels and bulk microcapillaries may be an admissible approach to accelerate this process, promoting overall pre-vascularization for long-term viability of constructs. However, the limited availability of bioinks that possess suitable characteristics that support both fabrication of complex architectures and formation of microcapillaries, remains a barrier to advancement in this space. In this study, gelatin-norbornene (Gel-NOR) is investigated as a vascular bioink with tailorable physico-mechanical properties, which promoted the self-assembly of human stromal and endothelial cells into microcapillaries, as well as being compatible with extrusion and lithography-based biofabrication modalities. Gel-NOR constructs containing self-assembled microcapillaries are successfully biofabricated with varying physical architecture (fiber diameter, spacing, and orientation). Both channel sizes and cell types affect the overall structural changes of the printed constructs, where cross-signaling between both human stromal and endothelial cells may be responsible for the reduction in open channel lumen observed over time. Overall, this work highlights an exciting three-way interplay between bioink formulation, construct design, and cell-mediated response that can be exploited towards engineering vascular tissues.

Strategies for inclusion of growth factors into 3D printed bone grafts.

There remains a critical need to develop new technologies and materials that can meet the demands of treating large bone defects. The advancement of 3-dimensional (3D) printing technologies has allowed the creation of personalized and customized bone grafts, with specific control in both macro- and micro-architecture, and desired mechanical properties. Nevertheless, the biomaterials used for the production of these bone grafts often possess poor biological properties. The incorporation of growth factors (GFs), which are the natural orchestrators of the physiological healing process, into 3D printed bone grafts, represents a promising strategy to achieve the bioactivity required to enhance bone regeneration. In this review, the possible strategies used to incorporate GFs to 3D printed constructs are presented with a specific focus on bone regeneration. In particular, the strengths and limitations of different methods, such as physical and chemical cross-linking, which are currently used to incorporate GFs to the engineered constructs are critically reviewed. Different strategies used to present one or more GFs to achieve simultaneous angiogenesis and vasculogenesis for enhanced bone regeneration are also covered in this review. In addition, the possibility of combining several manufacturing approaches to fabricate hybrid constructs, which better mimic the complexity of biological niches, is presented. Finally, the clinical relevance of these approaches and the future steps that should be taken are discussed.

Endochondral Bone Regeneration by Non-autologous Mesenchymal Stem Cells.

Mimicking endochondral bone formation is a promising strategy for bone regeneration. To become a successful therapy, the cell source is a crucial translational aspect. Typically, autologous cells are used. The use of non-autologous mesenchymal stromal cells (MSCs) represents an interesting alternative. Nevertheless, non-autologous, differentiated MSCs may trigger an undesired immune response, hampering bone regeneration. The aim of this study was to unravel the influence of the immune response on endochondral bone regeneration, when using xenogeneic (human) or allogeneic (Dark Agouti) MSCs. To this end, chondrogenically differentiated MSCs embedded in a collagen carrier were implanted in critical size femoral defects of immunocompetent Brown Norway rats. Control groups were included with syngeneic/autologous (Brown Norway) MSCs or a cell-free carrier. The amount of neo-bone formation was proportional to the degree of host-donor relatedness, as no full bridging of the defect was observed in the xenogeneic group whereas 2/8 and 7/7 bridges occurred in the allogeneic and the syngeneic group, respectively. One week post-implantation, the xenogeneic grafts were invaded by pro-inflammatory macrophages, T lymphocytes, which persisted after 12 weeks, and anti-human antibodies were developed. The immune response toward the allogeneic graft was comparable to the one evoked by the syngeneic implants, aside from an increased production of alloantibodies, which might be responsible for the more heterogeneous bone formation. Our results demonstrate for the first time the feasibility of using non-autologous MSC-derived chondrocytes to elicit endochondral bone regeneration in vivo. Nevertheless, the pronounced immune response and the limited bone formation observed in the xenogeneic group undermine the clinical relevance of this group. On the contrary, although further research on how to achieve robust bone formation with allogeneic cells is needed, they may represent an alternative to autologous transplantation.

Contrast enhanced computed tomography for real-time quantification of glycosaminoglycans in cartilage tissue engineered constructs.

Tissue engineering and regenerative medicine are two therapeutic strategies to treat, and to potentially cure, diseases affecting cartilaginous tissues, such as osteoarthritis and cartilage defects. Insights into the processes occurring during regeneration are essential to steer and inform development of the envisaged regenerative strategy, however tools are needed for longitudinal and quantitative monitoring of cartilage matrix components. In this study, we introduce a contrast-enhanced computed tomography (CECT)-based method using a cationic iodinated contrast agent (CA4+) for longitudinal quantification of glycosaminoglycans (GAG) in cartilage-engineered constructs. CA4+ concentration and scanning protocols were first optimized to ensure no cytotoxicity and a facile procedure with minimal radiation dose. Chondrocyte and mesenchymal stem cell pellets, containing different GAG content were generated and exposed to CA4+. The CA4+ content in the pellets, as determined by micro computed tomography, was plotted against GAG content, as measured by 1,9-dimethylmethylene blue analysis, and showed a high linear correlation. The established equation was used for longitudinal measurements of GAG content over 28 days of pellet culture. Importantly, this method did not adversely affect cell viability or chondrogenesis. Additionally, the CA4+ distribution accurately matched safranin-O staining on histological sections. Hence, we show proof-of-concept for the application of CECT, utilizing a positively charged contrast agent, for longitudinal and quantitative imaging of GAG distribution in cartilage tissue-engineered constructs. STATEMENT OF SIGNIFICANCE: Tissue engineering and regenerative medicine are promising therapeutic strategies for different joint pathologies such as cartilage defects or osteoarthritis. Currently, in vitro assessment on the quality and composition of the engineered cartilage mainly relies on destructive methods. Therefore, there is a need for the development of techniques that allow for longitudinal and quantitative imaging and monitoring of cartilage-engineered constructs. This work harnesses the electrostatic interactions between the negatively-charged glycosaminoglycans (GAGs) and a positively-charged contrast agent for longitudinal and non-destructive quantification of GAGs, providing valuable insight on GAG development and distribution in cartilage engineered constructs. Such technique can advance the development of regenerative strategies, not only by allowing continuous monitoring but also by serving as a pre-implantation screening tool.

Soft-molecular imprinted electrospun scaffolds to mimic specific biological tissues.

The fabrication of bioactive scaffolds able to mimic the in vivo cellular microenvironment is a challenge for regenerative medicine. The creation of sites for the selective binding of specific endogenous proteins represents an attractive strategy to fabricate scaffolds able to elicit specific cell response. Here, electrospinning (ESP) and soft-molecular imprinting (soft-MI) techniques were combined to fabricate a soft-molecular imprinted electrospun bioactive scaffold (SMIES) for tissue regeneration. Scaffolds functionalized using different proteins and growth factors (GFs) arranged onto the surface were designed, fabricated and validated with different polyesters, demonstrating the versatility of the developed approach. The scaffolds bound selectively each of the different proteins used, indicating that the soft-MI method allowed fabricating high affinity binding sites on ESP fibers compared to non-imprinted ones. The imprinting of ESP fibers with several GFs resulted in a significant effect on cell behavior. FGF-2 imprinted SMIES promoted cell proliferation and metabolic activity. BMP-2 and TGF-ß3 imprinted SMIES promoted cellular differentiation. These scaffolds hold the potential to be used in a cell-free approach to steer endogenous tissue regeneration in several regenerative medicine applications.

Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity.

Additive manufactured three-dimensional (3D) scaffolds with tailored surface topography constitute a clear advantage in tissue regeneration strategies to steer cell behavior. 3D fibrous scaffolds of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer presenting different fiber surface features were successfully fabricated by additive manufacturing combined with wet-spinning, in a single step, without any post-processing. The optimization of the processing parameters, mainly driven by different solvent/non-solvent combinations, led to four distinct scaffold types, with average surface roughness values ranging from 0.071 ± 0.012 µm to 1.950 ± 0.553 µm, average pore sizes in the x- and y-axis between 351.1 ± 33.6 µm and 396.1 ± 32.3 µm, in the z-axis between 36.5 ± 5.3 µm and 70.7 ± 8.8 µm, average fiber diameters between 69.4 ± 6.1 µm and 99.0 ± 9.4 µm, and porosity values ranging from 60.2 ± 0.8% to 71.7 ± 2.6%. Human mesenchymal stromal cells (hMSCs) cultured on these scaffolds adhered, proliferated, and produced endogenous extracellular matrix. The effect of surface roughness and topography on hMSCs differentiation was more evident for cells seeded at lower density, where the percentage of cells in direct contact with the surface was higher compared to more densely seeded scaffolds. Under osteogenic conditions, lower surface roughness values (0.227 ± 0.035 µm) had a synergistic effect on hMSCs behavior, while chondrogenesis was favored on rougher surfaces (1.950 ± 0.553 µm).

Toward mimicking the bone structure: design of novel hierarchical scaffolds with a tailored radial porosity gradient.

Guiding bone regeneration poses still unmet challenges due to several drawbacks of current standard treatments in the clinics. A possible solution may rely on the use of three-dimensional scaffolds with optimized structural properties in combination with human mesenchymal stem cells (hMSCs). Bone presents a radial gradient structure from the outside, where the cortical bone is more compact (porosity ranging from 5% to 10%), toward the inner part, where the cancellous bone is more porous (porosity ranging from 50% to 90%). Here, we present a new scaffold design with a built-in gradient in porosity, which approximate the radial bone structure. The pores of the outer ring were 500 µm, the ones in the middle zone were 750 µm and the inner part presented pores of 1000 µm. The porosity of each scaffold region resembled the gradient present in bone, with the outer ring having a porosity of 29.6% ± 5%, the middle and inner regions a porosity of 50.8% ± 8.1% and 77.6% ± 3.2% respectively. hMSCs behavior was analyzed in terms of growth, extracellular matrix deposition and differentiation toward the osteogenic lineage. A trend was displayed by the hMSCs residing in different zones of the gradient scaffolds after 7, 14 and 28 days of culture in mineralization medium. Osteogenic differentiation was influenced by pore size and location in scaffolds displaying a radial porosity gradient. Cell differentiation was confirmed by gene expression with upregulation of Runx2 and bone sialoprotein markers. Mineralization staining further confirmed the maturation of cell differentiation, as indicated by the presence of calcium and phosphate mineral deposits.

Surface energy and stiffness discrete gradients in additive manufactured scaffolds for osteochondral regeneration.

Swift progress in biofabrication technologies has enabled unprecedented advances in the application of developmental biology design criteria in three-dimensional scaffolds for regenerative medicine. Considering that tissues and organs in the human body develop following specific physico-chemical gradients, in this study, we hypothesized that additive manufacturing (AM) technologies would significantly aid in the construction of 3D scaffolds encompassing such gradients. Specifically, we considered surface energy and stiffness gradients and analyzed their effect on adult bone marrow derived mesenchymal stem cell differentiation into skeletal lineages. Discrete step-wise macroscopic gradients were obtained by sequentially depositing different biodegradable biomaterials in the AM process, namely poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymers. At the bulk level, PEOT/PBT homogeneous scaffolds supported a higher alkaline phosphatase (ALP) activity compared to PCL, PLA, and gradient scaffolds, respectively. All homogeneous biomaterial scaffolds supported also a significantly higher amount of glycosaminoglycans (GAGs) production compared to discrete gradient scaffolds. Interestingly, the analysis of the different material compartments revealed a specific contribution of PCL, PLA, and PEOT/PBT to surface energy gradients. Whereas PEOT/PBT regions were associated to significantly higher ALP activity, PLA regions correlated with significantly higher GAG production. These results show that cell activity could be influenced by the specific spatial distribution of different biomaterial chemistries in a 3D scaffold and that engineering surface energy discrete gradients could be considered as an appealing criterion to design scaffolds for osteochondral regeneration.