Bone Regeneration Engineering Research Articles

Hydrogen Sulfide Delivery to Enhance Bone Tissue Engineering Cell Survival

Though crucial for natural bone healing, local calcium ion (Ca2+) and phosphate ion (Pi) concentrations can exceed the cytotoxic limit leading to mitochondrial overload, oxidative stress, and cell death. For bone tissue engineering applications, H2S can be employed as a cytoprotective molecule to enhance mesenchymal stem cell (MSC) tolerance to cytotoxic Ca2+/Pi concentrations. Varied concentrations of sodium hydrogen sulfide (NaSH), a fast-releasing H2S donor, were applied to assess the influence of H2S on MSC proliferation. The results suggested a toxicity limit of 4 mM for NaSH and that 1 mM of NaSH could improve cell proliferation and differentiation in the presence of cytotoxic levels of Ca2+ (32 mM) and/or Pi (16 mM). To controllably deliver H2S over time, a novel donor molecule (thioglutamic acid—GluSH) was synthesized and evaluated for its H2S release profile. Excitingly, GluSH successfully maintained cytoprotective level of H2S over 7 days. Furthermore, MSCs exposed to cytotoxic Ca2+/Pi concentrations in the presence of GluSH were able to thrive and differentiate into osteoblasts. These findings suggest that the incorporation of a sustained H2S donor such as GluSH into CaP-based bone graft substitutes can facilitate considerable cytoprotection, making it an attractive option for complex bone regenerative engineering applications.

Bone Regeneration Facilitated by Autologous Bioscaffold Material: Liquid Phase of Concentrated Growth Factor with Dental Follicle Stem Cell Loading.

The application of bioengineering techniques for achieving bone regeneration in the oral environment is an increasingly prominent field. However, the clinical use of synthetic materials carries certain risks. The liquid phase of concentrated growth factor (LPCGF), as a biologically derived material, exhibits superior biocompatibility. In this study, LPCGF was employed as a tissue engineering scaffold, hosting dental follicle cells (DFCs) to facilitate bone regeneration. Both in vivo and in vitro experimental results demonstrate that this platform significantly enhances the expression of osteogenic markers in DFCs, such as alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and type I collagen (Col1a1). Simultaneously, it reduces the expression of inflammation-related genes, particularly interleukin-6 (IL-6) and interleukin-8 (IL-8), thereby alleviating the negative impact of the inflammatory microenvironment on DFCs. Further investigation into potential mechanisms reveals that this process is regulated over time by the WNT pathway. Our research results demonstrate that LPCGF, with its favorable physical characteristics, holds great potential as a scaffold. It can effectively carry DFCs, thereby providing an optimal initial environment for bone regeneration. Furthermore, LPCGF endeavors to closely mimic the mechanisms of bone healing post-trauma to facilitate bone formation. This offers new perspectives and insights into bone regeneration engineering.

Research Progress of Design Drugs and Composite Biomaterials in Bone Tissue Engineering.

Bone, like most organs, has the ability to heal naturally and can be repaired slowly when it is slightly injured. However, in the case of bone defects caused by diseases or large shocks, surgical intervention and treatment of bone substitutes are needed, and drugs are actively matched to promote osteogenesis or prevent infection. Oral administration or injection for systemic therapy is a common way of administration in clinic, although it is not suitable for the long treatment cycle of bone tissue, and the drugs cannot exert the greatest effect or even produce toxic and side effects. In order to solve this problem, the structure or carrier simulating natural bone tissue is constructed to control the loading or release of the preparation with osteogenic potential, thus accelerating the repair of bone defect. Bioactive materials provide potential advantages for bone tissue regeneration, such as physical support, cell coverage and growth factors. In this review, we discuss the application of bone scaffolds with different structural characteristics made of polymers, ceramics and other composite materials in bone regeneration engineering and drug release, and look forward to its prospect.

Oxygen-Generating Biomaterials for Translational Bone Regenerative Engineering.

Successful regeneration of critical-size defects remains one of the significant challenges in regenerative engineering. These large-scale bone defects are difficult to regenerate and are often reconstructed with matrices that do not provide adequate oxygen levels to stem cells involved in the regeneration process. Hypoxia-induced necrosis predominantly occurs in the center of large matrices since the host tissue's local vasculature fails to provide sufficient nutrients and oxygen. Indeed, utilizing oxygen-generating materials can overcome the central hypoxic region, induce tissue in-growth, and increase the quality of life for patients with extensive tissue damage. This article reviews recent advances in oxygen-generating biomaterials for translational bone regenerative engineering. We discussed different oxygen-releasing and delivery methods, fabrication methods for oxygen-releasing matrices, biology, oxygen's role in bone regeneration, and emerging new oxygen delivery methods that could potentially be used for bone regenerative engineering.

Processing of gelatine coated composite scaffolds based on magnesium and strontium doped hydroxyapatite and yttria-stabilized zirconium oxide

Limited bone bank capacity and risk of infection are some of the main drawbacks of autologous and allogenic grafts, giving rise to synthetic materials for bone tissue implants. The aim of this study was to process and evaluate the mechanical properties and bioactivity of magnesium and strontium doped hydroxyapatite (HAp) scaffolds and investigate the effect of adding zirconium oxide and gelatine coating the scaffolds. Doped nanosized hydroxyapatite powder was synthesized by the hydrothermal method and the scaffolds were made by the foam replica technique and sintered at different temperatures. Yttria-stabilized zirconium oxide (YSZ), synthesized by plasma technology, was used as reinforcement of calcium phosphate scaffolds. Element analysis, phase composition, morphology of the powders and microstructure of the scaffolds were investigated, as well as the compressive strength of the coated and uncoated scaffolds and bioactivity in simulated body fluid (SBF). A microporous structure was achieved with interconnected pores and bioactivity in SBF was confirmed in all cases. The best mechanical properties were given by the coated composite HAp/YSZ scaffolds, withstanding average stresses of over 1019 kPa. These results encourage the idea of use of these scaffolds in bone regenerative therapy and bone tissue engineering.

Hydroxyapatite-Tethered Peptide Hydrogel Promotes Osteogenesis.

Hydroxyapatite (HAp) as natural bone composition is highly osteoinductive. To harvest its osteoinductivity in bone regenerative engineering, the HAp-supporting hydrogel is urgently needed to minimize inhomogeneous aggregation of HAp. Here, we developed a HAp-stabilizing hydrogel based on peptide self-assembly. FmocFFRR was efficient for HAp-capping due to arginine-phosphate interaction. Tethering FmocFFRR on the HAp surface facilitated self-assembly to form FmocFFRR/HAp hybrid hydrogel, enabling stable dispersion of HAp in it. The molecular interactions between FmocFFRR and HAp particles were studied using microscopic and spectral characterizations. FmocFFRR/HAp hydrogel exhibited more enhanced mechanical properties than FmocFFRR. The biocompatibility of FmocFFRR/HAp hydrogel was verified using an ATP assay and live-dead staining assay. More importantly, FmocFFRR/HAp hydrogel not only enabled cell attachment on its surface, but also supported 3D cell culturing inside the hydrogel. Further, 3D culturing of MC3T3-E1 preosteoblasts inside FmocFFRR/HAp hydrogel significantly enhanced the expressions of osteogenesis markers, including alkaline phosphate (ALP), type-I collagen (COL1), and osteocalcin (OCN), demonstrating the promoting effect of osteoblast differentiation. These findings inspire its potential application in bone regenerative engineering.

Small molecule-mediated regenerative engineering for craniofacial and dentoalveolar bone.

The comprehensive reconstruction of extensive craniofacial and dentoalveolar defects remains a major clinical challenge to this day, especially in complex medical cases involving cancer, cranioplasty, and traumatic injury. Currently, osteogenic small molecule-based compounds have been explored extensively to repair and regenerate bone tissue because of their unique advantages. Over the past few years, a number of small molecules with the potential of craniofacial and periodontal bone tissue regeneration have been reported in literature. In this review, we discuss current progress using small molecules to regulate cranial and periodontal bone regeneration. Future directions of craniofacial bone regenerative engineering using the small molecule-based compounds will be discussed as well.

Ultra-low binder content 3D printed calcium phosphate graphene scaffolds as resorbable, osteoinductive matrices that support bone formation in vivo

Bone regenerative engineering could replace autografts; however, no synthetic material fulfills all design criteria. Nanocarbons incorporated into three-dimensional printed (3DP) matrices can improve properties, but incorporation is constrained to low wt%. Further, unmodified nanocarbons have limited osteogenic potential. Functionalization to calcium phosphate graphene (CaPG) imparts osteoinductivity and osteoconductivity, but loading into matrices remained limited. This work presents ultra-high content (90%), 3DP-CaPG matrices. 3DP-CaPG matrices are highly porous (95%), moderately stiff (3 MPa), and mechanically robust. In vitro, they are cytocompatible and induce osteogenic differentiation of human mesenchymal stem cells (hMSCs), indicated by alkaline phosphatase, mineralization, and COL1α1 expression. In vivo, bone regeneration was studied using a transgenic fluorescent-reporter mouse non-union calvarial defect model. 3DP-CaPG stimulates cellular ingrowth, retains donor cells, and induces osteogenic differentiation. Histology shows TRAP staining around struts, suggesting potential osteoclast activity. Apparent resorption of 3DP-CaPG was observed and presented no toxicity. 3DP-CaPG represents an advancement towards a synthetic bone regeneration matrix.

MiR-153-3p inhibited osteogenic differentiation of human DPSCs through CBFβ signaling.

Dental pulp stem cells (DPSCs) have multilineage differentiation potential and especially show a great foreground in bone regeneration engineering. The mechanism of osteogenic differentiation of DPSCs needs to be explored exactly. As a kind of endogenous and non-coding small RNAs, microRNAs (miRNAs) play an important role in many biological processes including osteogenic differentiation. However, the mechanism of miR-153-3p in osteogenic differentiation of DPSCs is still unknown. Core-binding factors-beta (CBFβ) is a non-DNA-binding factor that combines with the runt-related transcription factor family transcription factors to mediate their DNA-binding affinities, and plays a critical role in regulating osteogenic differentiation. In this study, we explored the mechanisms of miR-153-3p and CBFβ in DPSC osteogenesis. The expression of miR-153-3p and CBFβ was tested under the osteogenic condition, and the influence led by changing the expression of miR-153-3p or CBFβ had also been detected. A luciferase reporter assay confirmed that miR-153-3p directly targeted to CBFβ. The osteogenic markers, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and bone morphogenetic protein 2 (BMP2), were tested in protein level or mRNA level. ALP and Alizarin red staining were used to detect the osteoblast activity and mineral deposition. In osteogenic condition, the expressions of CBFβ and osteogenic markers were upregulated, whereas that of miR-153-3p was downregulated. miR-153-3p negatively regulated the osteogenic differentiation, and overexpression of CBFβ could offset the negative effect of miR-153-3p. Our findings provided a novel strategy for DPSC application in treatment of bone deficiencies and facilitated bone regeneration.

Advanced graphene ceramics and their future in bone regenerative engineering

AbstractThe outstanding properties of graphene materials rely on an exceptional two‐dimensional honeycombed lattice. The lattice allows for electrical, thermal, and mechanical reinforcement effects when applied to the ceramic matrix. The biocompatibility of the material allows for providing multifunctional bioceramics applications. However, the potential of graphene lies in its ability to be homogenously distributed as part of a ceramic matrix. Therefore, appropriate processing techniques are important for attaining desired graphene ceramic properties applicable for regenerative biomedical purposes. This article provides an inclusive review of the current knowledge of advanced graphene‐based ceramics for bone regenerative engineering. In this review, the opportunities and challenges in utilizing graphene materials in combination with ceramics suitable for applications in load‐bearing bone defects are discussed.

Pharmaceutical electrospinning and 3D printing scaffold design for bone regeneration.

Bone regenerative engineering provides a great platform for bone tissue regeneration covering cells, growth factors and other dynamic forces for fabricating scaffolds. Diversified biomaterials and their fabrication methods have emerged for fabricating patient specific bioactive scaffolds with controlled microstructures for bridging complex bone defects. The goal of this review is to summarize the points of scaffold design as well as applications for bone regeneration based on both electrospinning and 3D bioprinting. It first briefly introduces biological characteristics of bone regeneration and summarizes the applications of different types of material and the considerations for bone regeneration including polymers, ceramics, metals and composites. We then discuss electrospinning nanofibrous scaffold applied for the bone regenerative engineering with various properties, components and structures. Meanwhile, diverse design in the 3D bioprinting scaffolds for osteogenesis especially in the role of drug and bioactive factors delivery are assembled. Finally, we discuss challenges and future prospects in the development of electrospinning and 3D bioprinting for osteogenesis and prominent strategies and directions in future.

In Vivo Evaluation of the Regenerative Capability of Glycylglycine Ethyl Ester-Substituted Polyphosphazene and Poly(lactic-co-glycolic acid) Blends: A Rabbit Critical-Sized Bone Defect Model

In an effort to understand the biological capability of polyphosphazene-based polymers, three-dimensional biomimetic bone scaffolds were fabricated using the blends of poly[(glycine ethylglycinato)75(phenylphenoxy)25]phosphazene (PNGEGPhPh) and poly(lactic-co-glycolic acid) (PLGA), and an in vivo evaluation was performed in a rabbit critical-sized bone defect model. The matrices constructed from PNGEGPhPh-PLGA blends were surgically implanted into 15 mm critical-sized radial defects of the rabbits as structural templates for bone tissue regeneration. PLGA, which is the most commonly used synthetic bone graft substitute, was used as a control in this study. Radiological and histological analyses demonstrated that PNGEGPhPh-PLGA blends exhibited favorable in vivo biocompatibility and osteoconductivity, as the newly designed matrices allowed new bone formation to occur without adverse immunoreactions. The X-ray images of the blends showed higher levels of radiodensity than that of the pristine PLGA, indicating higher rates of new bone formation and regeneration. Micro-computed tomography quantification revealed that new bone volume fractions were significantly higher for the PNGEGPhPh-PLGA blends than for the PLGA controls after 4 weeks. The new bone volume increased linearly with increasing time points, with the new tissues observed throughout the defect area for the blend and only at the implant site's extremes for the PLGA control. Histologically, the polyphosphazene system appeared to show tissue responses and bone ingrowths superior to PLGA. By the end of the study, the defects with PNGEGPhPh-PLGA scaffolds exhibited evidence of effective bone tissue ingrowth and minimal inflammatory responses. Thus, polyphosphazene-containing biomaterials have excellent translational potential for use in bone regenerative engineering applications.

Cell Surface Glycoprotein CD24 Marks Bone Marrow-Derived Human Mesenchymal Stem/Stromal Cells with Reduced Proliferative and Differentiation Capacity In Vitro.

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) are fundamental to bone regenerative therapies, tissue engineering, and postmenopausal osteoporosis. Donor variation among patients, cell heterogeneity, and unpredictable capacity for differentiation reduce effectiveness of BMSCs for regenerative cell therapies. The cell surface glycoprotein CD24 exhibits the most prominent differential expression during osteogenic versus adipogenic differentiation of human BMSCs. Therefore, CD24 may represent a selective biomarker for subpopulations of BMSCs with increased osteoblastic potential. In undifferentiated human BMSCs, CD24 cell surface expression is variable among donors (range: 2%-10%) and increased by two to fourfold upon osteogenic differentiation. Strikingly, FACS sorted CD24pos cells exhibit delayed mineralization and reduced capacity for adipocyte differentiation. RNAseq analysis of CD24pos and CD24neg BMSCs identified a limited number of genes with increased expression in CD24pos cells that are associated with cell adhesion, motility, and extracellular matrix. Downregulated genes are associated with cell cycle regulation, and biological assays revealed that CD24pos cells have reduced proliferation. Hence, expression of the cell surface glycoprotein CD24 identifies a subpopulation of human BMSCs with reduced capacity for proliferation and extracellular matrix mineralization. Functional specialization among BMSCs populations may support their regenerative potential and therapeutic success by accommodating cell activities that promote skeletal tissue formation, homeostasis, and repair.

FEA evaluation of material stiffness changes for a polymer assisted 3D polycaprolactone/β-tricalcium phosphate scaffold in a mandibular defect reconstruction model

In bone regenerative engineering, the biomechanical performance of the scaffold at the bone-tissue interface is a key consideration. The evaluation of this parameter is a crucial step in designing, optimizing and manufacturing of bone substitute materials for clinical application, but is as yet comparatively unexplored. To this end, we utilized a novel polymer-assisted method to fabricate a three-dimensional (3D) Polycaprolactone/β-Tricalcium phosphate scaffold. The compressive modulus was measured and the data used to inform finite element analysis. The scaffolds achieved a maximum compressive moduli of 151 MPa, close to that of cortical bone. Further computational simulations were performed to determine the stresses and local scaffold adaptation profile, using data from computer tomography scans of the mandible. Local stresses were simulated based on the density changes in new bone forming in the scaffold at different stages of healing. The stress distribution in the mandible, scaffold center and scaffold interface were explored for a static load of 200 N, which corresponds to the load of adult mastication near the incisors. The analysis revealed that the maximum cross-sectional stress at the scaffold center and at the scaffold interface was 2.7 and 4.12 MPa respectively. The majority of the stress was localized in the bone of the mandible, with the scaffold bearing minimal loading at the start, but more over time as infiltration of more new bone progressed.

Graphene-Based Biomaterials for Bone Regenerative Engineering: A Comprehensive Review of the Field and Considerations Regarding Biocompatibility and Biodegradation.

Graphene and its derivatives have continued to garner worldwide interest due to their unique characteristics. Having expanded into biomedical applications, there have been efforts to employ their exceptional properties for the regeneration of different tissues, particularly bone. This article presents a comprehensive review on the usage of graphene-based materials for bone regenerative engineering. The graphene family of materials (GFMs) are used either alone or in combination with other biomaterials in the form of fillers in composites, coatings for both scaffolds and implants, or vehicles for the delivery of various signaling and therapeutic agents. The applications of the GFMs in each of these diverse areas are discussed and emphasis is placed on the characteristics of the GFMs that have implications in this regard. In tandem and of importance, this article evaluates the safety and biocompatibility of the GFMs and carefully elucidates how various factors influence the biocompatibility and biodegradability of this new class of nanomaterials. In conclusion, the challenges and opportunities regarding the use of the GFMs in regenerative engineering applications are discussed, and future perspectives for the developments in this field are proposed.

Ultra-small nanodots coated with oligopeptides providing highly negative charges to enhance osteogenic differentiation of hBMSCs better than osteogenic induction medium

For bone regenerative engineering, it is a promising method to form skeletal tissues differentiating from human bone morrow mesenchyme stem cells (hBMSCs). However, it is still a critical challenge to efficiently control ostogenesis and clearly reveal the influence factor. To this end, the fluorescent gold nanodots (Au NDs) with highly negative charges as osteogenic induction reagent are successfully synthesized, which display better than commercial osteogenic induction medium through the investigations of ALP activity (2.5 folds) and cytoskeleton staining (1.5 folds). Two kinds of oligopeptides with different bio-structures (cysteine, Cys and glutathione, GSH) are selected for providing surficial charges on Au NDs. It is revealed that Au-Cys with more negative charges (−51 mV) play better role than Au-GSH (−19 mV) in osteogenic differentiation, when both of them have same size (∼2 nm), sphere shape and show similar cell uptake amount. To explore deeply, osteogenesis related signaling pathways are monitored, revealing that the enhancement of osteogenic differentiation was through autophagy signaling pathway triggered by Au-Cys. And the promotion of highly negative charges in osteogenic differentiation was further proved via sliver nanodots (Ag NDs, Ag-Cys and Ag-GSH) and carbon nanodots (CDs, Cys-CDs and GSH-CDs). This work indicates part of insights during hBMSCs differentiation and provides a novel strategy in osteogenic differentiation process.

Repositioning Tacrolimus: Evaluation of the Effect of Short-Term Tacrolimus Treatment on Osteoprogenitor Cells and Primary Cells for Bone Regenerative Engineering.

Small-molecule-based bone regenerative engineering is an encouraging strategy for repair and regeneration of skeletal tissue. Using osteogenic small molecules for engineering bone tissue has several potential benefits over polypeptide-based approaches. Interestingly, hundreds of such small molecules possess the capability to promote osteogenesis, and several of these are already approved by the FDA for use in other applications, indicating their safety for human use. However, the need for their use at a high frequency and/or duration, due to their short half-life and nonspecificity, is still problematic. We, and others, have identified several non-FDA-approved small-molecule-based compounds that induce long-lasting osteogenic effects following short-term (<24 h) treatment. In this study, however, we have performed a proactive screen to investigate and compare the osteogenic effects of several preselected FDA-approved small-molecule drugs in vitro using osteoprogenitor MC3T3-E1 cells. Our results demonstrate that treatment with the small-molecule drug tacrolimus (FK-506) for 24 h significantly enhanced long-lasting osteogenic responses in both osteoprogenitor cells and primary cell cultures. In addition, we tested whether a short-term treatment with FK-506 is able to induce osteogenic differentiation of cells seeded on a polymeric scaffold in vitro. Using an osteogenic small molecule that has long-lasting effects despite a short duration of exposure to cells may alleviate the undesirable effects often seen with many osteogenic small molecules.

Novel osteogenic growth peptide C-terminal pentapeptide grafted poly(d,l-lactic acid) improves the proliferation and differentiation of osteoblasts: The potential bone regenerative biomaterial

Poly(d,l-lactic acid) (PDLLA) is widely used for bone regenerative engineering, because of its proven biocompatibility and biodegradability. However, the major limitation of PDLLA is its cell recognition and low hydrophilicity. The objective of this study was to develop a novel bioactive poly(d,l-lactic acid) tethered with osteogenic growth peptide (OGP), which has been confirmed as one of the important growth factors related to bone repair/regeneration. The biomimetic material modification methods were utilized that maleic anhydride-modified poly(d,l-lactic acid) (MPLA) as raw material, the active C-terminal pentapeptide OGP(10–14) were covalently grafted onto the side chain of MPLA through amide reaction using 1‑ethyl‑3‑(3‑dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N‑hydroxysuccinimide (NHS) as the condensing agent to produce a new biopolymer (OGP(10–14)-MPLA). The OGP(10–14)-MPLA were further characterized with the Fourier transform infrared spectrometry, amino acid analyzer, elementary analysis, X-ray photoelectron spectroscopy. The results revealed that OGP(10–14) was successfully modified MPLA and its coupling efficiency was 12.40%. The data from both contact angle and water absorption showed the better hydrophilicity of OGP(10–14)-MPLA, compared with MPLA. Also, we found that OGP(10–14)-MPLA could improve the proliferation, differentiation, and mineralization of osteoblasts, indicating that the novel OGP(10–14)-MPLA has the better biocompatibility and is more osteoinductive. In conclusion, the OGP(10–14) modified MPLA have the potential for bone regenerative engineering.

POLYMERIC BIOMATERIALS FOR SCAFFOLD-BASED BONE REGENERATIVE ENGINEERING.

Reconstruction of large bone defects resulting from trauma, neoplasm, or infection is a challenging problem in reconstructive surgery. The need for bone grafting has been increasing steadily partly because of our enhanced capability to salvage limbs after major bone loss. Engineered bone graft substitutes can have advantages such as lack of antigenicity, high availability, and varying properties depending on the applications chosen for use. These favorable attributes have contributed to the rise of scaffold-based polymeric tissue regeneration. Critical components in the scaffold-based polymeric regenerative engineering approach often include 1. The existence of biodegradable polymeric porous structures with properties selected to promote tissue regeneration and while providing appropriate mechanical support during tissue regeneration. 2. Cellular populations that can influence and enhance regeneration. 3. The use of growth and morphogenetic factors which can influence cellular migration, differentiation and tissue regeneration in vivo. Biodegradable polymers constitute an attractive class of biomaterials for the development of scaffolds due to their flexibility in chemistry and their ability to produce biocompatible degradation products. This paper presents an overview of polymeric scaffold-based bone tissue regeneration and reviews approaches as well as the particular roles of biodegradable polymers currently in use.

Nanofiber/Microsphere Hybrid Matrices In Vivo for Bone Regenerative Engineering: A Preliminary Report.

The demand for bone grafts has led to advances in regenerative engineering, a field at the intersection of advanced biomaterials, stem cell science, physics, developmental biology, and clinical translation. In this work, the authors evaluated a hybrid nanofiber/microsphere matrices both in vitro and in vivo for its ability to promote bone regeneration. Quantitative measures of cellular characteristics in vitro showed a higher fraction of marrow stromal cells with collagen promoter activity on hybrid matrices compared to control matrices (41% vs. 24%, p = 0.02). Control and hybrid matrices were then implanted for 6 weeks in calvarial defects of mice, and the animals received a single injection of calcein 1 day prior to sacrifice to visualize bone formation. Cryohistology of the undecalfied implants were evaluated for markers of bone mineralization, which revealed evidence of higher levels of bone tissue formation in hybrid matrices compared to controls. These data provide support that nanofiber-permeated, sintered, composite microsphere matrices may be a particularly useful matrix for the regenerative engineering of bone.