Chemotactic and Angiogenic Potential of Mineralized Collagen Scaffolds Functionalized with Naturally Occurring Bioactive Factor Mixtures to Stimulate Bone Regeneration

To develop cost-effective and efficient bone substitutes for improved regeneration of bone defects, heparin-modified mineralized collagen scaffolds were functionalized with concentrated, naturally occurring bioactive factor mixtures derived from adipose tissue, platelet-rich plasma and conditioned medium from a hypoxia-treated human bone marrow-derived mesenchymal stem cell line. Besides the analysis of the release kinetics of functionalized scaffolds, the bioactivity of the released bioactive factors was tested with regard to chemotaxis and angiogenic tube formation. Additionally, functionalized scaffolds were seeded with human bone marrow-derived mesenchymal stromal cells (hBM-MSC) and their osteogenic and angiogenic potential was investigated. The release of bioactive factors from the scaffolds was highest within the first 3 days. Bioactivity of the released factors could be confirmed for all bioactive factor mixtures by successful chemoattraction of hBM-MSC in a transwell assay as well as by the formation of prevascular structures in a 2D co-culture system of hBM-MSC and human umbilical vein endothelial cells. The cells seeded directly onto the functionalized scaffolds were able to express osteogenic markers and form tubular networks. In conclusion, heparin-modified mineralized collagen scaffolds could be successfully functionalized with naturally occurring bioactive factor mixtures promoting cell migration and vascularization.     

3D Printed SiOC(N) Ceramic Scaffolds for Bone Tissue Regeneration: Improved Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Bone tissue engineering has developed significantly in recent years as there has been increasing demand for bone substitutes due to trauma, cancer, arthritis, and infections. The scaffolds for bone regeneration need to be mechanically stable and have a 3D architecture with interconnected pores. With the advances in additive manufacturing technology, these requirements can be fulfilled by 3D printing scaffolds with controlled geometry and porosity using a low-cost multistep process. The scaffolds, however, must also be bioactive to promote the environment for the cells to regenerate into bone tissue. To determine if a low-cost 3D printing method for bespoke SiOC(N) porous structures can regenerate bone, these structures were tested for osteointegration potential by using human mesenchymal stem cells (hMSCs). This includes checking the general biocompatibilities under the osteogenic differentiation environment (cell proliferation and metabolism). Moreover, cell morphology was observed by confocal microscopy, and gene expressions on typical osteogenic markers at different stages for bone formation were determined by real-time PCR. The results of the study showed the pore size of the scaffolds had a significant impact on differentiation. A certain range of pore size could stimulate osteogenic differentiation, thus promoting bone regrowth and regeneration.     

Characterization of Osteogenesis and Chondrogenesis of Human Decellularized Allogeneic Bone with Mesenchymal Stem Cells Derived from Bone Marrow,Adipose Tissue,and Wharton’s Jelly

Allogeneic bone grafts are a promising material for bone implantation due to reduced operative trauma, reduced blood loss, and no donor-site morbidity. Although human decellularized allogeneic bone (hDCB) can be used to fill bone defects, the research of revitalizing hDCB blocks with human mesenchymal stem cells (hMSCs) for osteochondral regeneration is missing. The hMSCs derived from bone marrow, adipose tissue, and Wharton’s jelly (BMMSCs, ADMSCs, and UMSCs, respectively) are potential candidates for bone regeneration. This study characterized the potential of hDCB as a scaffold for osteogenesis and chondrogenesis of BMMSCs, ADMSCs, and UMSCs. The pore sizes and mechanical strength of hDCB were characterized. Cell survival and adhesion of hMSCs were investigated using MTT assay and F-actin staining. Alizarin Red S and Safranin O staining were conducted to demonstrate calcium deposition and proteoglycan production of hMSCs after osteogenic and chondrogenic differentiation, respectively. A RT-qPCR was performed to analyze the expression levels of osteogenic and chondrogenic markers in hMSCs. Results indicated that BMMSCs and ADMSCs exhibited higher osteogenic potential than UMSCs. Furthermore, ADMSCs and UMSCs had higher chondrogenic potential than BMMSCs. This study demonstrated that chondrogenic ADMSCs- or UMSCs-seeded hDCB might be potential osteochondral constructs for osteochondral regeneration.     

Robocasting and surface functionalization with highly bioactive glass of ZrO2 scaffolds for load bearing applications

This work is a proof of concept for making load bearing implants with osseointegration and bone bonding ability. Yttria-stabilized zirconia (YSZ) scaffolds with a percentage of macro porosity of about 70% were fabricated by robocasting. Although a maximum solids volume fraction of 50 vol.% could be achieved, the 3D-printing process revealed to be more reliable when using inks with 48 vol.% solids. The sintered porous structures exhibited an average compressive strength of ~236 MPa. After some preliminary coating experiments, an ethanol slurry of fine bioactive glass (BG) particles (10 wt.%) stabilized with polyvinylpyrrolidone was used to deposit a uniform surface coating onto the filaments, followed by glazing at 850°C. The functionalized scaffolds showed a relatively uniform surface coverage by the bioactive glass. The results of in vitro testing by immersing the scaffolds in simulated body fluid (SBF) showed remarkable morphological surface changes and an extensive deposition of hydroxyapatite layer. The overall results demonstrate the viability of producing porous YSZ scaffolds with excellent bioactivity, which are promising for bone tissue engineering under load bearing applications.     

Facilitation of Bone Healing Processes Based on the Developmental Function of Meox2 in Tooth Loss Lesion

In the present study, we examined the bone healing capacity of Meox2, a homeobox gene that plays essential roles in the differentiation of a range of developing tissues, and identified its putative function in palatogenesis. We applied the knocking down of Meox2 in human periodontal ligament fibroblasts to examine the osteogenic potential of Meox2. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of Meox2 knockdown for 1 and 2 weeks in bone healing processes. We examined the detailed histomorphological changes using Masson’s trichrome staining and micro-computed tomography evaluation. Moreover, we observed the localization patterns of various signaling molecules, including α-SMA, CK14, IL-1β, and MPO to examine the altered bone healing processes. Furthermore, we investigated the process of bone formation using immunohistochemistry of Osteocalcin and Runx2. On the basis of the results, we suggest that the knocking down of Meox2 via the activation of osteoblast and modulation of inflammation would be a plausible answer for bone regeneration as a gene therapy. Additionally, we propose that the purpose-dependent selection and application of developmental regulation genes are important for the functional regeneration of specific tissues and organs, where the pathological condition of tooth loss lesion would be.     

Biological Characteristics and Osteogenic Differentiation of Ovine Bone Marrow Derived Mesenchymal Stem Cells Stimulated with FGF-2 and BMP-2

Cell-based therapies using mesenchymal stem cells (MSCs) are a promising tool in bone tissue engineering. Bone regeneration with MSCs involves a series of molecular processes leading to the activation of the osteoinductive cascade supported by bioactive factors, including fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2). In this study, we examined the biological characteristics and osteogenic differentiation potential of sheep bone marrow MSCs (BM-MSCs) treated with 20 ng/mL of FGF-2 and 100 ng/mL BMP-2 in vitro. The biological properties of osteogenic-induced BM-MSCs were investigated by assessing their morphology, proliferation, phenotype, and cytokine secretory profile. The osteogenic differentiation was characterized by Alizarin Red S staining, immunofluorescent staining of osteocalcin and collagen type I, and expression levels of genetic markers of osteogenesis. The results demonstrated that BM-MSCs treated with FGF-2 and BMP-2 maintained their primary MSC properties and improved their osteogenic differentiation capacity, as confirmed by increased expression of osteocalcin and collagen type I and upregulation of osteogenic-related gene markers BMP-2, Runx2, osterix, collagen type I, osteocalcin, and osteopontin. Furthermore, sheep BM-MSCs produced a variety of bioactive factors involved in osteogenesis, and supplementation of the culture medium with FGF-2 and BMP-2 affected the secretome profile of the cells. The results suggest that sheep osteogenic-induced BM-MSCs may be used as a cellular therapy to study bone repair in the preclinical large animal model.     

Biomimetic Polycaprolactone-Graphene Oxide Composites for 3D Printing Bone Scaffolds

Bone shows a radial gradient architecture with the exterior densified cortical bone and the interior porous cancellous bone. However, previous studies presented uniform designs for bone scaffolds that do not mimic natural bone's gradient structure. Hence, mimicking native bone structures is still challenging in bone tissue engineering. In this study, a novel biomimetic bone scaffold with Haversian channels is designed, which approximates mimicking the native bone structure. Also, the influence of adding graphene oxide (GO) to polycaprolactone (PCL)-based scaffolds are investigated by preparing PCL/GO composite ink containing 0.25% and 0.75% GO and then 3D printing scaffolds by an extrusion-based machine. Scanning electron microscopy (SEM) is used for morphological analysis. SEM reveals good printability and interconnected pore structure. The contact angle test shows that wettability reinforces with the increase of GO content. The mechanical behavior of the scaffolds under compression is examined numerically and experimentally. The results indicate that incorporation of GO can affect bone scaffolds' Young's modulus and von Mises stress distribution. Moreover, the biodegradation rates accelerate in the PCL/GO scaffolds. Biological characterizations, such as cell growth, viability, and attachment, are performed utilizing osteoblast cells. Compared to pure PCL, an enhancement is observed in cell viability in the PCL/GO scaffolds.     

Biohybrid Bovine Bone Matrix for Controlled Release of Mesenchymal Stem/Stromal Cell Lyosecretome: A Device for Bone Regeneration

SmartBone^® (SB) is a biohybrid bone substitute advantageously proposed as a class III medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic, and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed. SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell (MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs (38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other hand, the process was still present, but tissue formation was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless, SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β.     

Effect of Morphological Characteristics and Biomineralization of 3D-Printed Gelatin/Hyaluronic Acid/Hydroxyapatite Composite Scaffolds on Bone Tissue Regeneration

The use of porous three-dimensional (3D) composite scaffolds has attracted great attention in bone tissue engineering applications because they closely simulate the major features of the natural extracellular matrix (ECM) of bone. This study aimed to prepare biomimetic composite scaffolds via a simple 3D printing of gelatin/hyaluronic acid (HA)/hydroxyapatite (HAp) and subsequent biomineralization for improved bone tissue regeneration. The resulting scaffolds exhibited uniform structure and homogeneous pore distribution. In addition, the microstructures of the composite scaffolds showed an ECM-mimetic structure with a wrinkled internal surface and a porous hierarchical architecture. The results of bioactivity assays proved that the morphological characteristics and biomineralization of the composite scaffolds influenced cell proliferation and osteogenic differentiation. In particular, the biomineralized gelatin/HA/HAp composite scaffolds with double-layer staggered orthogonal (GEHA20-ZZS) and double-layer alternative structure (GEHA20-45S) showed higher bioactivity than other scaffolds. According to these results, biomineralization has a great influence on the biological activity of cells. Hence, the biomineralized composite scaffolds can be used as new bone scaffolds in bone regeneration.     

99mTc-HDP Labeling—A Non-Destructive Method for Real-Time Surveillance of the Osteogenic Differentiation Potential of hMSC during Ongoing Cell Cultures

99-Metastabil Technetium (^99mTc) is a radiopharmaceutical widely used in skeletal scintigraphy. Recent publications show it can also be used to determine the osteogenic potential of human mesenchymal stem cells (hMSCs) by binding to hydroxyapatite formed during bone tissue engineering. This field lacks non-destructive methods to track live osteogenic differentiation of hMSCs. However, no data about the uptake kinetics of ^99mTc and its effect on osteogenesis of hMSCs have been published yet. We therefore evaluated the saturation time of ^99mTc by incubating hMSC cultures for different periods, and the saturation concentration by using different amounts of ^99mTc activity for incubation. The influence of ^99mTc on osteogenic potential of hMSCs was then evaluated by labeling a continuous hMSC culture three times over the course of 3 weeks, and comparing the findings to cultures labeled once. Our findings show that ^99mTc saturation time is less than 0.25 h, and saturation concentration is between 750 and 1000 MBq. Repeated exposure to γ-radiation emitted by ^99mTc had no negative effects on hMSC cultures. These new insights can be used to make this highly promising method broadly available to support researchers in the field of bone tissue engineering using this method to track and evaluate, in real-time, the osteogenic differentiation of hMSC, without any negative influence on the cell viability, or their osteogenic differentiation potential.     

The Marine Polysaccharide Ulvan Confers Potent Osteoinductive Capacity to PCL-Based Scaffolds for Bone Tissue Engineering Applications

Hybrid composites of synthetic and natural polymers represent materials of choice for bone tissue engineering. Ulvan, a biologically active marine sulfated polysaccharide, is attracting great interest in the development of novel biomedical scaffolds due to recent reports on its osteoinductive properties. Herein, a series of hybrid polycaprolactone scaffolds containing ulvan either alone or in blends with κ-carrageenan and chondroitin sulfate was prepared and characterized. The impact of the preparation methodology and the polysaccharide composition on their morphology, as well as on their mechanical, thermal, water uptake and porosity properties was determined, while their osteoinductive potential was investigated through the evaluation of cell adhesion, viability, and osteogenic differentiation of seeded human adipose-derived mesenchymal stem cells. The results verified the osteoinductive ability of ulvan, showing that its incorporation into the polycaprolactone matrix efficiently promoted cell attachment and viability, thus confirming its potential in the development of biomedical scaffolds for bone tissue regeneration applications.     

Cryogenic 3D Printing of w/o Pickering Emulsions Containing Bifunctional Drugs for Producing Hierarchically Porous Bone Tissue Engineering Scaffolds with Antibacterial Capability

How to fabricate bone tissue engineering scaffolds with excellent antibacterial and bone regeneration ability has attracted increasing attention. Herein, we produced a hierarchical porous β-tricalcium phosphate (β-TCP)/poly(lactic-co-glycolic acid)-polycaprolactone composite bone tissue engineering scaffold containing tetracycline hydrochloride (TCH) through a micro-extrusion-based cryogenic 3D printing of Pickering emulsion inks, in which the hydrophobic silica (h-SiO₂) nanoparticles were used as emulsifiers to stabilize composite Pickering emulsion inks. Hierarchically porous scaffolds with desirable antibacterial properties and bone-forming ability were obtained. Grid scaffolds with a macroscopic pore size of 250.03 ± 75.88 μm and a large number of secondary micropores with a diameter of 24.70 ± 15.56 μm can be fabricated through cryogenic 3D printing, followed by freeze-drying treatment, whereas the grid structure of scaffolds printed or dried at room temperature was discontinuous, and fewer micropores could be observed on the strut surface. Moreover, the impartment of β-TCP in scaffolds changed the shape and density of the micropores but endowed the scaffold with better osteoconductivity. Scaffolds loaded with TCH had excellent antibacterial properties and could effectively promote the adhesion, expansion, proliferation, and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells afterward. The scaffolds loaded with TCH could realize the strategy to “kill bacteria first, then induce osteogenesis”. Such hierarchically porous scaffolds with abundant micropores, excellent antibacterial property, and improved bone-forming ability display great prospects in treating bone defects with infection.     

Early Bone Healing on Hydroxyapatite-Coated and Chemically-Modified Hydrophilic Implant Surfaces in an Ovine Model

Implant topography affects early peri-implant bone healing by changing the osteoconduction rate in the surrounding biological environment. Implant surfaces have been designed to promote faster and stronger bone formation for rapid and stable prosthesis loading. Early peri-implant bone healing has been observed with a sandblasted, acid-etched implant that was chemically modified to be hydrophilic (cmSLA). The present study investigates whether early peri-implant bone healing extends to a rough surface implant with a high crystalline hydroxyapatite surface (TSV MP-1 HA). Three implants were randomly placed in porous trabecular bone within both medial femoral condyles of 10 sheep. Early peri-implant bone stability was measured at 3- and 6-weeks healing time following implant insertion. Results indicated a similar implant stability quotient between the implants at insertion and over time. The significant increase over time of reverse torque values with respect to insertion torque (p < 0.001) did not differ between the implants. However, the bone-to-implant contact of TSV MP-1 HA was significantly higher than that of cmSLA implants at 6 weeks (p < 0.01). These data validate previous findings of a hydrophilic implant surface and extend the observation of early osseointegration to a rough surface implant in porous trabecular bone.     

Evaluating Oxygen Tensions Related to Bone Marrow and Matrix for MSC Differentiation in 2D and 3D Biomimetic Lamellar Scaffolds

The physiological O₂ microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O₂, OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O₂. At 21% O₂, we measured significant increases in ultimate tensile strength (p < 0.0001) and Young’s modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O₂ hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties.     

Development of Collagen/Demineralized Bone Powder Scaffolds and Periosteum-Derived Cells for Bone Tissue Engineering Application

The aim of this study was to investigate physical and biological properties of collagen (COL) and demineralized bone powder (DBP) scaffolds for bone tissue engineering. DBP was prepared and divided into three groups, based on various particle sizes: 75–125 μm, 125–250 μm, and 250–500 μm. DBP was homogeneously mixed with type I collagen and three-dimensional scaffolds were constructed, applying chemical crosslinking and lyophilization. Upon culture with human periosteum-derived cells (PD cells), osteogenic differentiation of PD cells was investigated using alkaline phosphatase (ALP) activity and calcium assay kits. The physical properties of the COL/DBP scaffolds were obviously different from COL scaffolds, irrespective of the size of DBP. In addition, PD cells cultured with COL scaffolds showed significantly higher cell adhesion and proliferation than those with COL/DBP scaffolds. In contrast, COL/DBP scaffolds exhibited greater osteoinductive potential than COL scaffolds. The PD cells with COL/DBP scaffolds possessed higher ALP activity than those with COL scaffolds. PD cells cultured with COL/DBP scaffolds with 250–500 μm particle size yielded the maximum calcium deposition. In conclusion, PD cells cultured on the scaffolds could exhibit osteoinductive potential. The composite scaffold of COL/DBP with 250–500 μm particle size could be considered a potential bone tissue engineering implant.     

Exosome: A Novel Approach to Stimulate Bone Regeneration through Regulation of Osteogenesis and Angiogenesis

The clinical need for effective bone regeneration therapy remains in huge demands. However, the current “gold standard” treatments of autologous and allogeneic bone grafts may result in various complications. Furthermore, safety considerations of biomaterials and cell-based treatment require further clarification. Therefore, developing new therapies with stronger osteogenic potential and a lower incidence of complications is worthwhile. Recently, exosomes, small vesicles of endocytic origin, have attracted attention in bone regeneration field. The vesicles travel between cells and deliver functional cargoes, such as proteins and RNAs, thereby regulating targeted cells differentiation, commitment, function, and proliferation. Much evidence has demonstrated the important roles of exosomes in osteogenesis both in vitro and in vivo. In this review, we summarize the properties, origins and biogenesis of exosomes, and the recent reports using exosomes to regulate osteogenesis and promote bone regeneration.     

Osteogenic Potential of Mesenchymal Stem Cells from Adipose Tissue,Bone Marrow and Hair Follicle Outer Root Sheath in a 3D Crosslinked Gelatin-Based Hydrogel

Bone transplantation is regarded as the preferred therapy to treat a variety of bone defects. Autologous bone tissue is often lacking at the source, and the mesenchymal stem cells (MSCs) responsible for bone repair mechanisms are extracted by invasive procedures. This study explores the potential of autologous mesenchymal stem cells derived from the hair follicle outer root sheath (MSCORS). We demonstrated that MSCORS have a remarkable capacity to differentiate in vitro towards the osteogenic lineage. Indeed, when combined with a novel gelatin-based hydrogel called Osteogel, they provided additional osteoinductive cues in vitro that may pave the way for future application in bone regeneration. MSCORS were also compared to MSCs from adipose tissue (ADMSC) and bone marrow (BMMSC) in a 3D Osteogel model. We analyzed gel plasticity, cell phenotype, cell viability, and differentiation capacity towards the osteogenic lineage by measuring alkaline phosphatase (ALP) activity, calcium deposition, and specific gene expression. The novel injectable hydrogel filled an irregularly shaped lesion in a porcine wound model displaying high plasticity. MSCORS in Osteogel showed a higher osteo-commitment in terms of calcium deposition and expression dynamics of OCN, BMP2, and PPARG when compared to ADMSC and BMMSC, whilst displaying comparable cell viability and ALP activity. In conclusion, autologous MSCORS combined with our novel gelatin-based hydrogel displayed a high capacity for differentiation towards the osteogenic lineage and are acquired by non-invasive procedures, therefore qualifying as a suitable and expandable novel approach in the field of bone regeneration therapy.