Co-culture of smooth muscle cells and endothelial cells on three-dimensional bioprinted polycaprolactone scaffolds for cavernosal tissue engineering.

PURPOSE: In vitro evaluation of polycaprolactone (PCL) scaffolds fabricated by a three-dimensional (3D) printing technique for tissue engineering applications in the corpus cavernosum. MATERIALS AND METHODS: PCL scaffolds were fabricated by use of a 3 D bioprinting system. The 3D-printed scaffolds had interconnected structures for cell ingrowth. Human aortic smooth muscle cells (haSMCs) were seeded on the scaffold and cultured for 5 days, and then human umbilical vein endothelial cells (HUVECs) were also added on the scaffolds and co-cultured with haSMCs for up to 7 days. The ability of these scaffolds to support the growth of HUVECs and haSMCs was investigated in vitro. 3 D strand-deposited scaffolds were characterized by scanning electron microscopy (SEM) images and porosity measurement. RESULTS: SEM images showed the surface of the PCL scaffolds to be well covered by HUVECs and haSMCs. Immunofluorescent staining of α-flk1 and α-smooth muscle actin on the HUVECs and haSMCs seeded scaffolds confirmed that the cells remained viable and proliferated throughout the time course of the culture. CONCLUSION: 3 D bioprinting of a PCL scaffold is feasible for co-culturing of HUVECs and haSMCs. This was a preliminary study to investigate the possibility of fabrication of tissue-engineered corpus cavernosum.

Characterization and preparation of bio-tubular scaffolds for fabricating artificial vascular grafts by combining electrospinning and a 3D printing system.

The last decade has seen artificial blood vessels composed of natural polymer nanofibers grafted into human bodies to facilitate the recovery of damaged blood vessels. However, electrospun nanofibers (ENs) of biocompatible materials such as chitosan (CTS) suffer from poor mechanical properties. This study describes the design and fabrication of artificial blood vessels composed of a blend of CTS and PCL ENs and coated with PCL strands using rapid prototyping technology. The resulting tubular vessels exhibited excellent mechanical properties and showed that this process may be useful for vascular reconstruction.

Tissue-engineered tracheal reconstruction using three-dimensionally printed artificial tracheal graft: preliminary report.

Three-dimensional printing has come into the spotlight in the realm of tissue engineering. We intended to evaluate the plausibility of 3D-printed (3DP) scaffold coated with mesenchymal stem cells (MSCs) seeded in fibrin for the repair of partial tracheal defects. MSCs from rabbit bone marrow were expanded and cultured. A half-pipe-shaped 3DP polycaprolactone scaffold was coated with the MSCs seeded in fibrin. The half-pipe tracheal graft was implanted on a 10 × 10-mm artificial tracheal defect in four rabbits. Four and eight weeks after the operation, the reconstructed sites were evaluated bronchoscopically, radiologically, histologically, and functionally. None of the four rabbits showed any sign of respiratory distress. Endoscopic examination and computed tomography showed successful reconstruction of trachea without any collapse or blockage. The replaced tracheas were completely covered with regenerated respiratory mucosa. Histologic analysis showed that the implanted 3DP tracheal grafts were successfully integrated with the adjacent trachea without disruption or granulation tissue formation. Neo-cartilage formation inside the implanted graft was sufficient to maintain the patency of the reconstructed trachea. Scanning electron microscope examination confirmed the regeneration of the cilia, and beating frequency of regenerated cilia was not different from those of the normal adjacent mucosa. The shape and function of reconstructed trachea using 3DP scaffold coated with MSCs seeded in fibrin were restored successfully without any graft rejection.

BMP-2-immobilized PCL 3D printing scaffold with a leaf-stacked structure as a physically and biologically activated bone graft.

Although three-dimensional (3D) printing techniques are used to mimic macro- and micro-structures as well as multi-structural human tissues in tissue engineering, efficient target tissue regeneration requires bioactive 3D printing scaffolds. In this study, we developed a bone morphogenetic protein-2 (BMP-2)-immobilized polycaprolactone (PCL) 3D printing scaffold with leaf-stacked structure (LSS) (3D-PLSS-BMP) as a bioactive patient-tailored bone graft. The unique LSS was introduced on the strand surface of the scaffold via heating/cooling in tetraglycol without significant deterioration in physical properties. The BMP-2 adsorbed on3D-PLSS-BMPwas continuously released from LSS over a period of 32 d. The LSS can be a microtopographical cue for improved focal cell adhesion, proliferation, and osteogenic differentiation.In vitrocell culture andin vivoanimal studies demonstrated the biological (bioactive BMP-2) and physical (microrough structure) mechanisms of3D-PLSS-BMPfor accelerated bone regeneration. Thus, bioactive molecule-immobilized 3D printing scaffold with LSS represents a promising physically and biologically activated bone graft as well as an advanced tool for widespread application in clinical and research fields.

Decellularized matrix bioink with gelatin methacrylate for simultaneous improvements in printability and biofunctionality.

Gelatin methacrylate (GelMA) bioink has been widely used in bioprinting because it is a printable and biocompatible biomaterial. However, it is difficult to print GelMA bioink without any temperature control because it has a thermally-sensitive rheological property. Therefore, in this study, we developed a temperature-controlled printing system in real time without affecting the viability of the cells encapsulated in the bioink. In addition, a skin-derived decellularized extracellular matrix (SdECM) was printed with GelMA to better mimic the native tissue environment compared with solely using GelMA bioink with the enhancement of structural stability. The temperature setting accuracy was calculated to be 98.58 ± 1.8 % for the module and 99.48 ± 1.33 % for the plate from 5 °C to 37 °C. The group of the temperature of the module at 10 °C and the plate at 20 °C have 93.84 % cell viability with the printable range in the printability window. In particular, the cell viability and proliferation were increased in the encapsulated fibroblasts in the GelMA/SdECM bioink, relative to the GelMA bioink, with a morphology that significantly spread for seven days. The gene expression and growth factors related to skin tissue regeneration were relatively upregulated with SdECM components. In the bioprinting process, the rheological properties of the GelMA/SdECM bioink were successfully adjusted in real time to increase printability, and the native skin tissue mimicked components providing tissue-specific biofunctions to the encapsulated cells. The developed bioprinting strategies and bioinks could support future studies related to the skin tissue reconstruction, regeneration, and other medical applications using the bioprinting process.

Combination therapy with BMP-2 and BMSCs enhances bone healing efficacy of PCL scaffold fabricated using the 3D plotting system in a large segmental defect model.

The three-dimensional (3D) plotting system is a rapidly-developing scaffold fabrication method for bone tissue engineering. It yields a highly porous and inter-connective structure without the use of cytotoxic solvents. However, the therapeutic effects of a scaffold fabricated using the 3D plotting system in a large segmental defect model have not yet been demonstrated. We have tested two hypotheses: whether the bone healing efficacy of scaffold fabricated using the 3D plotting system would be enhanced by bone marrow-derived mesenchymal stem cell (BMSC) transplantation; and whether the combination of bone morphogenetic protein-2 (BMP-2) administration and BMSC transplantation onto the scaffold would act synergistically to enhance bone regeneration in a large segmental defect model. The use of the combined therapy did increase bone regeneration further as compared to that with monotherapy in large segmental bone defects.

An osteogenic bioink composed of alginate, cellulose nanofibrils, and polydopamine nanoparticles for 3D bioprinting and bone tissue engineering.

Bioprinting is an emerging technology for manufacturing cell-laden three-dimensional (3D) scaffolds, which are used to fabricate complex 3D constructs and provide specific microenvironments for supporting cell growth and differentiation. The development of bioinks with appropriate printability and specific bioactivities is crucial for bioprinting and tissue engineering applications, including bone tissue regeneration. Therefore, to produce functional bioinks for osteoblast printing and bone tissue formation, we formulated various nanocomposite hydrogel-based bioinks using natural and biocompatible biomaterials (i.e., alginate, tempo-oxidized cellulose nanofibrils (TOCNF), and polydopamine nanoparticles (PDANPs)). Rheological studies and printability tests revealed that bioinks containing 1.5% alginate and 1.5% TOCNF in the presence or absence of PDANP (0.5%) are suitable for 3D printing. Furthermore, in vitro studies of 3D-printed osteoblast-laden scaffolds indicated that the 0.5% PDANP-incorporated bioink induced significant osteogenesis. Overall, the bioink consisting of alginate, TOCNF, and PDANPs exhibited excellent printability and bioactivity (i.e., osteogenesis).

Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering.

For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL), those from PCL and hydroxyapatite (HA), and those from PCL/HA and with a shifted pattern structure (PCL/HA/SP scaffold). Shifted pattern structure was fabricated to increase the cell attachment/adhesion. The PCL/HA/SP scaffold had a lower compressive modulus than PCL and PCL/HA scaffold. However, it has a better cell attachment than the scaffolds without a shifted pattern. MTT assay and alkaline phosphatase activity results for the PCL/HA/SP scaffolds were significantly enhanced compared to the results for the PCL and PCL/HA scaffolds. According to their degree of cell proliferation/differentiation, the scaffolds were in the following order: PCL/HA/SP > PCL/HA > PCL. These 3D scaffolds will be applicable for tissue engineering based on unique plotting system.

Surface modification of a three-dimensional polycaprolactone scaffold by polydopamine, biomineralization, and BMP-2 immobilization for potential bone tissue applications.

Three-dimensional (3D) bioprinting is a free-form fabrication technique enabling fine feature control for tissue engineering applications. Especially, 3D scaffolds capable of supporting cell attachment, proliferation, and osteogenic differentiation are a prerequisite for bone tissue regeneration. Herein, we elaborated this approach to produce a 3D polycaprolactone (PCL) scaffold with long-term osteogenic activity. Specifically, we coated polydopamine (PDA) on 3D PCL scaffolds, subsequently deposited hydroxyapatite (HA) nanoparticles via biomimetic mineralization, and finally immobilized bone morphogenetic protein-2 (BMP-2). Material properties were characterized and compared with various 3D scaffolds, including PCL, PDA-coated PCL (PCL/PDA), and PDA-coated and HA-deposited PCL (PCL/PDA/HA). In vitro cell culture studies with osteoblasts revealed that the PCL/PDA/HA scaffolds immobilized with BMP-2 showed long-term retention of BMP-2 (for up to 21 days) and significantly increased osteoblast proliferation and osteogenic differentiation, as evidenced by metabolic activity, alkaline phosphatase activity, and calcium deposition. We believe that this multifunctional osteogenic 3D scaffold will be useful for bone tissue engineering applications.

Experimental investigation of esophageal reconstruction with electrospun polyurethane nanofiber and 3D printing polycaprolactone scaffolds using a rat model.

BACKGROUND: We evaluated the outcome of esophageal reconstructions using tissue-engineered scaffolds. METHOD: Partial esophageal defects were reconstructed with the following scaffolds; animals were grouped (n = 7 per group) as follows: (a) normal rats; (b) rats implanted with three-dimensional printing (3DP) polycaprolactone (PCL) scaffolds; (c) with human adipose-derived mesenchymal stem cell (ADSC)-seeded 3DP PCL scaffolds; (d) with polyurethane (PU)-nanofiber(Nf) scaffolds; and (e) with ADSC-seeded PU-Nf scaffolds. RESULTS: The esophageal defects were successfully repaired; however, muscle regeneration was greater in the 3DP PCL + ADSC groups than in the PU-Nf + ADSC groups (P < .001). Regeneration of the epithelium was greater in PU-Nf and PU-Nf + ADSC groups than in the 3DP PCL and 3DP PCL + ADSC groups (P < .001). CONCLUSION: A tendency for more re-epithelization was observed with the PU-Nf scaffolds, while more muscle regeneration was achieved with the 3DP PCL scaffolds.

Prevascularized Tracheal Scaffolds Using the Platysma Flap for Enhanced Tracheal Regeneration.

OBJECTIVES: One of the greatest hurdles in tracheal tissue engineering is insufficient vascularization, which leads to delayed mucosal regeneration, inflammation, and restenosis. This study investigated whether a prevascularized segmental tracheal substitute using platysma can enhance tracheal mucosal regeneration. METHODS: Three-dimensional (3D) printed scaffolds with (group M) or without (group S) Matrigel coating were implanted under the feeding vessels of the platysma in New Zealand White rabbits (n = 3) to induce vascularization. After 1 or 2 weeks, tracheal defects were created and vascularized scaffolds with feeders of the platysma were transplanted as rotational flaps. As controls, scaffolds with or without Matrigel coating was transplanted into a tracheal defect without prevascularization. Airway patency and epithelization were examined using a rigid bronchoscope every 2 weeks. Surviving animals were euthanized at 24 weeks, and microcomputed tomography and histological evaluation were performed. RESULTS: Animals with 2 weeks of prevascularization showed longer survival than animals with 0 or 1 weeks of prevascularization regardless of the Matrigel coating. Wider airway patency was observed in group M than group S. Group M showed migration of epithelium over the scaffold from 4 weeks after transplantation and complete coverage with epithelium at 12 weeks, whereas group S showed migration of the epithelium from 14 weeks and incomplete coverage with epithelium even at 24 weeks. CONCLUSION: This two-step method, utilizing the platysma as an in vivo bioreactor, may be a promising approach to achieve long-term survival and enhanced luminal patency. Matrigel coating on the scaffold had a synergistic effect on epithelial regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1732-1740, 2021.

Three-dimensionally printed polycaprolactone/beta-tricalcium phosphate scaffold was more effective as an rhBMP-2 carrier for new bone formation than polycaprolactone alone.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) has been widely used in bone tissue engineering to enhance bone regeneration because of its osteogenic inductivity. However, clinical outcomes can vary depending on the scaffold materials used to deliver rhBMP-2. In this study, 3D-printed scaffolds with a ratio of 1:1 polycaprolactone and beta-tricalcium phosphate (PCL/T50) were applied as carriers for rhBMP-2 in mandibular bone defect models in dog models. Before in vivo application, in vitro experiments were conducted. Preosteoblast proliferation was not significantly different between scaffolds made of PCL/T50 and polycaprolactone alone (PCL/T0) regardless of rhBMP-2 delivery. However, PCL/T50 showed an increased level of the alkaline phosphatase activity and mineralization assay when rhBMP-2 was delivered. In in vivo, the newly formed bone volume of the PCL/T50 group was significantly increased compared with that of the PCL/T0 scaffolds regardless of rhBMP-2 delivery. Histological examination showed that PCL/T50 with rhBMP-2 produced significantly greater amounts of newly bone formation than PCL/T0 with rhBMP-2. The quantities of scaffold remaining were lower in the PCL/T50 group than in the PCL/T0 group, although it was not significantly different. In conclusion, PCL/T50 scaffolds were advantageous for rhBMP-2 delivery as well as for maintaining space for bone formation in mandibular bone defects.

Self-crosslinkable hyaluronate-based hydrogels as a soft tissue filler.

With increasing interest in aging and skin care, the use of fillers to increase the volume of soft tissue volume is increasing globally. However, the side effects caused by the residual chemical crosslinking agents present in these fillers limit the effective application of commercialized filler products. Therefore, the development of a novel crosslinking system with a non-toxic chemical crosslinking agent is required to overcome the limitations of commercial hyaluronate (HA)-based fillers. In this paper, a new injectable hydrogel with enhanced mechanical properties, tissue adhesion, injectability, and biocompatibility is reported. The HA derivatives modified with catechol groups (HA-DA) were crosslinked by self-oxidation under in vivo physiological conditions (pH 7.4) without chemical crosslinkers to form hydrogels, which can be further accelerated by the dissolved oxygen in the body. The fabricated HA-DA filler showed excellent mechanical properties and could be easily injected with a low injection force. Further, the HA-DA filler stably attached to the injection site due to the tissue adhesion properties of the catechol groups, thus leading to an improved displacement stability. In addition, the HA-DA filler showed excellent cell viability, cell proliferation, and biocompatibility. Therefore, the HA-DA hydrogel is a novel soft tissue filler with great potential to overcome the limitations of commercial soft tissue fillers.

Bio-plotted hydrogel scaffold with core and sheath strand-enhancing mechanical and biological properties for tissue regeneration.

Three-dimensional bio-plotted scaffolds constructed from encapsulated biomaterials or so-called "bio-inks" have received much attention for tissue regeneration applications, as advances in this technology have enabled more precise control over the scaffold structure. As a base material of bio-ink, sodium alginate (SA) has been used extensively because it provides suitable biocompatibility and printability in terms of creating a biomimetic environment for cell growth, even though it has limited cell-binding moiety and relatively weak mechanical properties. To improve the mechanical and biological properties of SA, herein, we introduce a strategy using hydroxyapatite (HA) nanoparticles and a core/sheath plotting (CSP) process. By characterizing the rheological and chemical properties and printability of SA and SA/HA-blended inks, we successfully fabricated bio-scaffolds using CSP. In particular, the mechanical properties of the scaffold were enhanced with increasing concentrations of HA particles and SA hydrogel. Specifically, HA particles blended with the SA hydrogel of core strands enhanced the biological properties of the scaffold by supporting the sheath part of the strand encapsulating osteoblast-like cells. Based on these results, the proposed scaffold design shows great promise for bone-tissue regeneration and engineering applications.

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

3D Printing of Bone-Mimetic Scaffold Composed of Gelatin/ß-Tri-Calcium Phosphate for Bone Tissue Engineering.

3D printed scaffolds composed of gelatin and ß-tri-calcium phosphate (ß-TCP) as a biomimetic bone material are fabricated, thereby providing an environment appropriate for bone regeneration. The Ca2+ in ß-TCP and COO- in gelatin form a stable electrostatic interaction, and the composite scaffold shows suitable rheological properties for bioprinting. The gelatin/ß-TCP scaffold is crosslinked with glutaraldehyde vapor and unreacted aldehyde groups which can cause toxicity to cells is removed by a glycine washing. The stable binding of the hydrogel is revealed as a result of FTIR and degradation rate. It is confirmed that the composite scaffold has compressive strength similar to that of cancellous bone and 60 wt% ß-TCP groups containing 40 wt% gelatin have good cellular activity with preosteoblasts. Also, in the animal experiments, the gelatin/ß-TCP scaffold confirms to induce bone formation without any inflammatory responses. This study suggests that these fabricated scaffolds can serve as a potential bone substitute for bone regeneration.

Graphene oxide/alginate composites as novel bioinks for three-dimensional mesenchymal stem cell printing and bone regeneration applications.

Three-dimensional (3D) cell printing is a versatile technique enabling the creation of 3D constructs containing hydrogel and cells in the desired shape or pattern. Bioinks exhibiting appropriate mechanical properties and biological activities to support cell growth and/or differentiation toward a specific lineage play critical roles in 3D cell printing and tissue engineering applications. Herein, we explored alginate/graphene oxide (GO) composites as bioinks for their potential to improve printability, structural stability, and osteogenic activities for osteogenic tissue engineering applications. The addition of GO (0.05-1.0 mg mL-1) to 3% alginate significantly enhanced the printing performances of the alginate bioink. In addition, mesenchymal stem cells (MSCs) printed with alginate/GO showed good proliferation and higher survival in an oxidative stress environment. The 3D scaffolds printed with MSCs and alginate/GO demonstrated significantly enhanced osteogenic differentiation compared with those printed with MSCs and alginate. Overall, a bioink of 3% alginate and 0.5 mg mL-1 GO showed the most balanced characteristics in terms of printability, structural stability, and osteogenic induction of the printed MSCs. Alginate/GO composite bioinks will be useful for bioprinting research for various tissue engineering applications.

Vascular endothelial growth factor immobilized on mussel-inspired three-dimensional bilayered scaffold for artificial vascular graft application: In vitro and in vivo evaluations.

Currently, there is a great clinical demand for biocompatible and robust tissue-engineered tubular scaffolds for use as artificial vascular graft materials. Despite considerable research on vascular scaffolds, there has still been only limited development of scaffold materials possessing both sufficient mechanical strengths and biological effects for vascular application. In this work, we designed a mechanically robust, bilayered scaffold and manufactured it by combining electrospinning (ELSP) and three-dimensional (3D) printing techniques. This material was coated with polydopamine (PDA) and vascular endothelial growth factor (VEGF) was grafted directly on the scaffold surface to induce potent angiogenic activity. We confirmed that the coated-PDA layer was evenly deposited on the bare polycaprolactone (PCL) scaffold and could enable abundant VEGF immobilization with enhanced hydrophilicity. The VEGF immobilized porous tubular scaffold was well prepared without mechanical weakness induced by surface modification steps. During in vitro and in vivo testing, VEGF immobilized scaffolds elicited markedly enhanced vascular cell proliferation and angiogenic differentiation, as compared to non-treated groups. These results demonstrate that the developed scaffolds may represent an innovative paradigm in vascular tissue engineering by inducing angiogenesis as a means of remodeling and healing vascular defects for use in restorative procedures.

Tissue-Engineered Esophagus via Bioreactor Cultivation for Circumferential Esophageal Reconstruction.

The use of biomaterials for circumferential esophageal repair is technically challenging in a rat model, and an optimal scaffold implantation technique with nutritional support is essential. The purpose of this study was to investigate the effects of three-dimensional printed esophageal grafts and bioreactor cultivation on muscle regeneration and reepithelialization from circumferential esophageal defects in a rat model. Here, we designed an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a two-layered tubular scaffold and mesenchymal stem cell-based bioreactor system. The graft was verified by the performance comparison with an omentum-cultured esophageal scaffold. We also applied a new surgical anastomosis technique and a thyroid gland flap over the implanted scaffold to improve graft survival. Although no regenerated mucosal layer was observed around the implants of the control group, histological examination of the regenerative esophagi along the scaffold revealed that the bioreactor system and omentum-cultured groups showed more than 80% of the mucosal regeneration without a fistula. The regenerated tissues showed that the integration of the esophageal scaffold and its native esophageal tissue was intact and were covered with layers of stratified squamous epithelium with several newly developed blood vessels. Therefore, this study describes a novel approach for circumferential esophageal reconstruction. Impact Statement In vivo functional esophageal reconstruction remains challenging due to anastomosis site leakage and necrosis of the implanted scaffold in a circumferential esophageal defect. Therefore, it is necessary to develop a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle without leakage of the esophageal anastomosis. In this study, we proposed an intriguing strategy that combines a mesenchymal stem cell-seeded tubular scaffold with a bioreactor system for esophageal reconstruction and introduced a new surgical anastomosis technique with the thyroid gland flap over the implanted scaffold to improve graft survival. We believe that this system should be a powerful platform for circumferential replacement of the esophagus in a rat model.

In situ gold nanoparticle growth on polydopamine-coated 3D-printed scaffolds improves osteogenic differentiation for bone tissue engineering applications: in vitro and in vivo studies.

In this study, we designed scaffolds coated with gold nanoparticles (GNPs) grown on a polydopamine (PDA) coating of a three-dimensional (3D) printed polycaprolactone (PCL) scaffold. Our results demonstrated that the scaffolds developed here may represent an innovative paradigm in bone tissue engineering by inducing osteogenesis as a means of remodeling and healing bone defects.