Porous Hydroxyapatite Scaffolds Research Articles

Rapid fabrication of mechanical-improved hydroxyapatite scaffolds from the microwave sintering with homogenous incorporated nanosilver particles

In this study, the optimization of the dual-frequency microwave sintering furnace in the replica method was investigated for the rapid synthesis of Porous hydroxyapatite scaffolds (PHS). It was found that by controlling the microwave sintering condition, it is possible to produce PHS with promising abilities for bone tissue engineering applications.

Effect of porogen agent on the size, shape, and structure of porous hydroxyapatite scaffolds

Effect of porogen agent on the size, shape, and structure of porous hydroxyapatite scaffolds

A Porous Fluoride-Substituted Bovine-Derived Hydroxyapatite Scaffold Constructed for Applications in Bone Tissue Regeneration.

Hydroxyapatite is widely used in bone implantation because of its similar mineral composition to natural bone, allowing it to serve as a biocompatible osteoconductive support. A bovine-derived hydroxyapatite (BHA) scaffold was developed through an array of defatting and deproteinization procedures. The BHA scaffold was substituted with fluoride ions using a modified sol-gel method to produce a bovine-derived fluorapatite (BFA) scaffold. Fourier-transform infrared spectroscopy and X-ray diffraction analysis showed that fluoride ions were successfully substituted into the BHA lattice. According to energy dispersive X-ray analysis, the main inorganic phases contained calcium and phosphorus with a fluoride ratio of ~1-2 wt%. Scanning electron microscopy presented a natural microporous architecture for the BFA scaffold with pore sizes ranging from ~200-600 μm. The BHA scaffold was chemically stable and showed sustained degradation in simulated-body fluid. Young's modulus and yield strength were superior in the BFA scaffold to BHA. In vitro cell culture studies showed that the BFA was biocompatible, supporting the proliferative growth of Saos-2 osteoblast cells and exhibiting osteoinductive features. This unique technique of producing hydroxyapatite from bovine bone with the intent of producing high performance biomedically targeted materials could be used to improve bone repair.

Digital light processing bio-scaffolds of hydroxyapatite ceramic foams with multi-level pores using Pickering emulsions as the feedstock

To prepare bio-scaffolds with multi-stage pores, excellent mechanical and biological properties to meet the requirements of personalized bone repair, digital light processing using Pickering emulsion as paste has been proposed here firstly for porous hydroxyapatite scaffolds with complex shape and fine microstructure. It is found that dispersant content, solid loading and oil to water ratio of Pickering emulsions play vital roles on the controlling of pore morphology and pore size distribution of final hydroxyapatite ceramic foams. The multi-stage pores consist of millimeter-scale pores designed by 3D models and micro-pores below 10 µm derived from emulsion droplets. Due to the integration of hierarchical pores at different scale levels and micropores, hydroxyapatite ceramic foams possess outstanding compressive strength of 4.46–8.03 MPa at porosity of 76.59-72.96%. In vitro cell experiments illustrate that rat bone mesenchymal stem cells can spread and adhere to bio-scaffolds, promoting cell proliferation effectively and further verifying their good biocompatibility.

3D printing of customized bioceramics for promoting bone tissue regeneration by regulating sympathetic nerve behavior.

Numerous studies have shown that there are multiple neural activities involved in the process of bone resorption and bone regeneration, and promoting osteogenesis by promoting neural network reconstruction is an effective strategy for repairing critical size bone defects. However, traumatic bone defects often cause activation of the sympathetic nervous system (SNS) in the damaged area, releasing excess catecholamines (CAs), resulting in a decrease in the rate of bone formation. Herein, a 3D-printed scaffold loaded with propranolol (PRN) is proposed to reduce CA concentrations in bone defect areas and promote bone regeneration through drug release. For this purpose, PRN-loaded methacrylated gelatin (GelMA) microspheres were mixed with low-concentration GelMA and perfused into a 3D-printed porous hydroxyapatite (HAp) scaffold. By releasing PRN, which can block β-adrenergic receptors, it hinders the activation of sympathetic nerves and inhibits the release of excess CA by the SNS. At the same time, the composite scaffold recruits bone marrow mesenchymal stem cells (BMSCs) and promotes the differentiation of BMSCs in the direction of osteoblasts, which effectively promotes bone regeneration in the rabbit femoral condyle defect model. The results of the study showed that the release of PRN from the composite scaffold could effectively hinder the activation of sympathetic nerves and promote bone regeneration, providing a new strategy for the treatment of bone defects.

Freeze casting of hydroxyapatite-titania composites for bone substitutes.

Hydroxyapatite (HA) is commonly used as a bone substitute material, but it lacks mechanical strength when compared to native bone tissues. To improve the efficacy of HA as a bone substitute by improving the mechanical strength and cell growth attributes, porous composite scaffolds of HA and titania (HA-TiO2 ) were fabricated through a freeze-casting process. Three different compositions by weight percent, 25-75 HA-TiO2 , 50-50 HA-TiO2 , and 75-25 HA-TiO2 , were custom-made for testing. After sintering at 1250°C, these composite scaffolds exhibited improved mechanical properties compared to porous HA scaffolds. Substrate mixing was observed, which helped reduce crystal size and introduced new phases such as β-TCP and CaTiO3 , which also led to improved mechanical properties. The composition of 50-50 HA-TiO2 had the highest ultimate compressive strength of 3.12 ± 0.36 MPa and elastic modulus 63.29 ± 28.75 MPa. Human osteoblast cell proliferation assay also increased on all three different compositions when compared to porous HA at 14 days. These results highlight the potential of freeze casting composites for the fabrication of bone substitutes, which provide enhanced mechanical strength and biocompatibility while maintaining porosity.

Mechanical properties of additive-manufactured hydroxyapatite porous scaffolds and follow-up of damage process under compression loading

In this work, the mechanical response and the damage process during compressive loading tests of hydroxyapatite scaffolds processed by stereolithography and designed for biomedical applications were investigated. Damage is followed by acoustic emission monitoring, Scanning Electron Microscopy observations at different loading stages and by In-situ X-ray tomography. A significant and progressive damage evolution is observed at stress value superior to 25 MPa. The cracks responsible for the damage are initiated from the edge of the sample and propagate parallel to the applied force, which explains the small variation of the Young's modulus, initially of 85 GPa, during loading/unloading sequences. Finite element analysis confirmed that the stress is non-uniform and maximum at the edge of the sample, which confirms the scenario of damage evolution.

A novel porous hydroxyapatite scaffold (pHAMG) enhances angiogenesis and osteogenesis around dental implants by regulating the immune microenvironment.

The purpose was to evaluate whether a novel porous hydroxyapatite (HA) scaffold with a 25-30-µm groove structure (pHAMG) may improve bone osteogenesis, angiogenesis, and bone integration of titanium dental implants in animal models. The pHAMG was prepared by chemical precipitation method and its elemental composition and crystal structure were evaluated. The ability of the scaffolds to induce ectopic osteogenesis and the ability of scaffolds combined with titanium dental implants to induce orthotopic peri-implant angiogenesis, osteogenesis, and osteointegration were tested after implantation into the femur muscle pocket in rats and the mandibular defects in beagle dogs, respectively. The elemental composition was evaluated by SEM-EDS; the expression of the relevant osteogenic/inflammation marker and the anti-/pro-inflammation markers was evaluated by immunostaining and immunofluorescence, respectively. In animal experiments with ectopic and peri-implant osteogenesis, pHAMG resulted in significantly larger neovascularization by hematoxylin-eosin staining, as well as deposition of collagen fibers by Masson staining than HA. Meanwhile, microgrooves in pHAMG upregulate more bone morphogenetic protein (BMP) 2 and interleukin-4 (IL-4) and -10 (IL-10) and downregulate more IL-1β and tumor necrosis factor-α (TNF-α) than that in HA. The pHAMG showed greater expression of arginase (Arg)-1 and lower expression of inducible nitric oxide synthase (iNOS) than HA. The novel pHAMG can better repair bone defects in ectopic and orthotopic model. It also transfers macrophages to anti-inflammatory phenotypes, promoting angiogenic and osteogenesis in scaffolds, and bone integration in implants. The novel pHAMG induce greater osteogenesis and angiogenesis which could be utilized in the clinical treatment.

Effect of different HA/β-TCP coated 3D printed bioceramic scaffolds on repairing large bone defects in rabbits.

Treatment of large segmental bone defects is still a major clinical challenge, and bone grafting is the main method. The development of novel bone graft substitutes will help solve this problem. Porous bioceramics hydroxyapatite (HA) scaffolds coated with different ratios of HA/β-tricalcium phosphate (β-TCP) were prepared by 3D printing. The scaffolds were sampled and tested in large segmental bone defect rabbit models. X-ray, micro-computed tomography (CT), hematoxylin and eosin (HE) staining, Van-Gieson staining, and type I collagen staining were performed to find the best scaffolds for large segmental bone defect treatment. The average length, diameter, compressive strength, and porosity of the bioceramics scaffolds were 15.05 ± 0.10mm, 4.98 ± 0.06mm, 11.11 ± 0.77MPa, and 54.26 ± 5.38%, respectively. Postoperative lateral radiographs suggested the scaffold group got better bone healing and stability than the blank group. Micro-CT showed new bones grew into the scaffold from the two ends of the fracture along the scaffold and finally achieved bony union. The new bone volume around the scaffolds suggested the 3:7 HA/β-TCP-coated bioceramic scaffolds were more favorable for the healing of large segmental bone defects. The results of HE, Van-Gieson, and type I collagen staining also suggested more new bone formation in 3:7 HA/β-TCP-coated bioceramic scaffolds. 3:7 HA/β-TCP-coated porous bioceramics scaffolds are more conducive to the repair of large bone defects in rabbits. The results of this study can provide some reference and theoretical support in this area.

Experimental and numerical investigations for compressive behavior of porous hydroxyapatite bone scaffold fabricated via freeze-gel casting method

Artificial bone scaffolds with a porosity of ≥64% were manufactured using hydroxyapatite (HA). The Freeze-gel Casting method using tert-butyl alcohol (TBA) as a solvent was adopted to maintain proper mechanical strength. The manufactured HA scaffold had excellent internal connectivity owing to the continuous connection of the long columnar pores and arrangement of the constant pore walls. For the HA scaffold, compression tests at different strain rates under uniaxially compressed loads were performed, and the compressive behavior according to the strain rate was analyzed. A maximum yield stress of 2–3 MPa was observed, and the overall compressive behavior was divided into elastic, plateau, and densification sections, which were different from the brittleness behavior of ordinary ceramics and similar to that of polymer materials. The compressive behavior of the HA scaffold was successfully simulated by applying the Frank–Brockman–Zairi constitutive model used to simulate elasto-viscoplastic material behavior. Seven material parameters belonging to the constitutive equation were proposed for the HA scaffold by adopting a deterministic approach for material parameter identification. Models can be developed from methods for simulating the mechanical behavior of a porous ceramic scaffold to predict the long-term mechanical behavior of the scaffold inserted in vivo, which may help in bone defects recovery.

A Novel Porous Butyryl Chitin-Animal Derived Hydroxyapatite Composite Scaffold for Cranial Bone Defect Repair.

Bone defects, a common orthopedic problem in clinical practice, are a serious threat to human health. As alternative materials to autologous bone grafts, synthetic cell-free functionalized scaffolds have been the focus of recent research in designing scaffolds for bone tissue engineering. Butyryl chitin (BC) is a derivative of chitin (CT) with improved solubility. It has good biocompatibility, but few studies have investigated its use in bone repair. In this study, BC was successfully synthesized with a degree of substitution of 2.1. BC films were prepared using the cast film method and showed strong tensile strength (47.8 ± 4.54 N) and hydrophobicity (86.4 ± 2.46°), which was favorable for mineral deposition. An in vitro cytological assay confirmed the excellent cell attachment and cytocompatibility of the BC film; meanwhile, in vivo degradation indicated the good biocompatibility of BC. Hydroxyapatite (HA), extracted from bovine cancellous bone, had good cytocompatibility and osteogenic induction activity for the mouse osteoblast cell line MC3T3-E1. With the aim of combining the advantages of BC and HA, a BC-HA composite scaffold, with a good pore structure and mechanical strength, was prepared by physical mixing. Administered into skull defects of rats, the scaffolds showed perfect bone-binding performance and effective structural support, and significantly promoted the regeneration of new bone. These results prove that the BC-HA porous scaffold is a successful bone tissue engineering scaffold and has strong potential to be further developed as a substitute for bone transplantation.

Preparation and characterization of porous hydroxyapatite reinforced with hydroxyapatite whiskers

In this study, porous hydroxyapatite scaffolds reinforced with whiskers were fabricated. HAp whiskers were produced by the molten salt synthesis method using 5 different salt-HAp mixtures. The mixtures placed in alumina crucibles were heated to 900 oC with a heating rate of 5 oC/min in a muffle furnace and kept at this temperature for 2 hours and then cooled in the furnace. To clean the HAP crystals formed in the alumina crucibles from the reaction residues, they were washed many times with distilled water heated to 100 oC and filtered. HAp whiskers were mixed with HAp powder in certain proportions, and porous HAp reinforced with HAp whiskers were fabricated by using the sponge replica method. The synthesized whiskers and the scaffold structures were characterized by Scanning Electron Microscopy-Energy Dispersive X-Ray Spectroscopy, X-ray diffractometry, Compression test, and porosity measurement method.XRD analysis of synthesized whiskers confirmed the presence of HAp. SEM images showed interconnected pores in the samples, with pore sizes larger than 100µm. The compressive strengths of the samples were calculated from the stress-strain plateau average. The highest and the lowest compressive stress were calculated as 0,125 and 0,050 MPa respectively. The maximum and minimum porosities of the samples reinforced with whiskers were found to be 67,56% and 61,92%, respectively.

Application of the neuropeptide NPVF to enhance angiogenesis and osteogenesis in bone regeneration

The brain-bone regulatory system regulates skeletal homeostasis via bioactive neuropeptides, yet the underlying mechanism remains elusive. Here, we report the role of the neuropeptide VF (NPVF, VPNLPQRF-NH2) in enhancing both angiogenesis and osteogenesis in a rat skeletal system and the potential pathways involved. An in vitro study revealed that NPVF not only promotes migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) by activating NPFFR1, which leads to upregulation of miR-181c-3p and downregulation of Argonaute1 (AGO1), but also mediates osteogenic differentiation of bone mesenchymal stem cells (BMSCs) via the Wnt/β-catenin signaling pathway. To improve the stability and bioavailability and thus efficacy of NPVF as a promoter of in vivo bone regeneration, we genetically engineered amyloid-NPVF-fusion proteins and utilized them as self-assembling nanofiber coatings to treat bone defects in a rat calvarial defect model. We found that a porous hydroxyapatite scaffold loaded with the NPVF peptide-fused amyloid coating substantially enhanced angiogenesis and site-specific fresh bone in-growth when implanted in calvarial defects. Taken together, our work uncovered a previously undefined crosstalk between the brain and bone by unveiling the role of NPVF in bone tissue and demonstrated a viable method for promoting bone tissue repairs based upon self-assembling NPVF-containing protein coatings.

Halloysite nanoclay reinforced hydroxyapatite porous scaffold for hard tissue regeneration

Hydroxyapatite (HAP), a natural constituent of bone tissue is commonly used for the clinical treatment of bone defects due to its similar structure with bone and excellent biocompatibility. However, the processing exertion, poor osteoinductive capability and poor mechanical strength of HAP needs further addressing for its immense implementation in tissue engineering. Different approaches have been reported to escalate the mechanical hardness and osteogenic potential of HAP. In the present work, halloysite nanoclay (HNC) and sericin protein were used for better mechanical and osteogenic properties, respectively. Halloysite nanoclay (HNC, 1.0–4.0%) was used to reinforce hydroxyapatite (HAP) and the mechanical strength of nanocomposite scaffolds were evaluated. After surface modification of nanocomposite scaffolds with 1.0% silk sericin protein; physical properties like microstructure, porosity, swelling ratio and degradation rate were evaluated. Cell morphology, cytocompatibility and alkaline phosphatase (ALP) activity were assessed using MG 63 osteoblast cell lines. HAP reinforced with 4% HNC (HAP@4) showed a significant increase (199 MPa) in young modulus as compared to pure HAP. HAP reinforced with 2% HNC (HAP@2) and 4% HNC (HAP@4) showed a significant decrease in porosity as well as degradation rate than pure HAP but no significant difference was observed in swelling ratio. The scanning electron microscope (SEM) images of the scaffolds showed porous architecture. Remarkably, the incorporation of HNC in HAP enhanced the cytocompatibility as well as ALP activity in comparison to pure HAP. Overall, these results suggested that halloysite nanoclay reinforced HAP scaffold could be an auspicious alternative for bone tissue regeneration.

Zn-doped chitosan/alginate multilayer coatings on porous hydroxyapatite scaffold with osteogenic and antibacterial properties.

Porous hydroxyapatite (HA) scaffolds prepared by three-dimensional (3D) printing have wide application prospects owing to personalized structural design and excellent biocompatibility. However, the lack of antimicrobial properties limits its widespread use. In this study, a porous ceramic scaffold was fabricated by digital light processing (DLP) method. The multilayer chitosan/alginate composite coatings prepared by layer-by-layer method were applied to scaffolds and Zn2+ was doped into coatings in the form of ion crosslinking. The chemical composition and morphology of coatings were characterized by scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS). Energy dispersive spectroscopy (EDS) analysis demonstrated that Zn2+ was uniformly distributed in the coating. Besides, the compressive strength of coated scaffolds (11.52 ± 0.3 MPa) was slightly improved compared with that of bare scaffolds (10.42 ± 0.56 MPa). The result of soaking experiment indicated that coated scaffolds exhibited delayed degradation. In vitro experiments demonstrated that within the limits of concentration, a higher Zn content in the coating has a stronger capacity to promote cell adhesion, proliferation and differentiation. Although excessive release of Zn2+ led to cytotoxicity, it presented a stronger antibacterial effect against Escherichia coli (99.4%) and Staphylococcus aureus (93%).

Bone Regeneration Guided by a Magnetized Scaffold in an Ovine Defect Model

The reconstruction of large segmental defects still represents a critical issue in the orthopedic field. The use of functionalized scaffolds able to create a magnetic environment is a fascinating option to guide the onset of regenerative processes. In the present study, a porous hydroxyapatite scaffold, incorporating superparamagnetic Fe3O4 nanoparticles (MNPs), was implanted in a critical bone defect realized in sheep metatarsus. Superparamagnetic nanoparticles functionalized with hyperbranched poly(epsilon-Lysine) peptides and physically complexed with vascular endothelial growth factor (VEGF) where injected in situ to penetrate the magnetic scaffold. The scaffold was fixed with cylindrical permanent NdFeB magnets implanted proximally, and the magnetic forces generated by the magnets enabled the capture of the injected nanoparticles forming a VEGF gradient in its porosity. After 16 weeks, histomorphometric measurements were performed to quantify bone growth and bone-to-implant contact, while the mechanical properties of regenerated bone via an atomic force microscopy (AFM) analysis were investigated. The results showed increased bone regeneration at the magnetized interface; this regeneration was higher in the VEGF-MNP-treated group, while the nanomechanical behavior of the tissue was similar to the pattern of the magnetic field distribution. This new approach provides insights into the ability of magnetic technologies to stimulate bone formation, improving bone/scaffold interaction.

Controlling the hierarchical microstructure of bioceramic scaffolds by 3D printing of emulsion inks

Mechanical and biological properties constitute the most fundamental requirements for bone tissue engineering (BTE) scaffolds. Nonetheless, existing fabrication strategies find it difficult to prepare highly porous BTE scaffolds for improved biological properties while also preserving sufficient mechanical properties that are compatible with the natural bone. Inspired by the hierarchical porous materials in Nature, hierarchical porous BTE scaffolds can achieve a combination of superior mechanical efficiency and biological functions. With this in mind, this study reports the fabrication of hierarchical porous hydroxyapatite (hpHA) scaffolds by 3D printing of emulsion inks. The scaffolds exhibit high porosity up to 73.7%, featuring 3D printed macropores of 300 – 400 µm and emulsion templated microporosity of < 20 µm. Via formulation of the emulsion inks, such as varying the oil volume and adding Pluronic® F-127, this process demonstrates effective control of the microporosity and pore morphology of the scaffolds. The scaffolds are mechanically compatible with the natural cancellous bone, with compressive strength in the range of 1.41 – 7.84 MPa and Young’s modulus of 57.3 – 304 MPa. Furthermore, the elastic admissible strain (EAS) and specific energy absorption (SEA) of the hpHA scaffolds can be increased up to 4.4% and 1.22 kJ/kg, respectively, indicating greatly enhanced mechanical performances owing to the hierarchical porous structure. Meanwhile, improved cell attachment, spreading and proliferation are observed in these scaffolds with their additional microporosity. Hence, the hpHA scaffolds in this study show great potential in BTE applications, and the reported process of 3D printing of emulsion inks is a promising fabrication strategy for further optimization of highly porous BTE scaffolds.

Surface Functionalization of Hydroxyapatite Scaffolds with MgAlEu-LDH Nanosheets for High-Performance Bone Regeneration.

Although artificial bone repair scaffolds, such as titanium alloy, bioactive glass, and hydroxyapatite (HAp), have been widely used for treatment of large-size bone defects or serious bone destruction, they normally exhibit unsatisfied bone repair efficiency because of their weak osteogenic and angiogenesis performance as well as poor cell crawling and adhesion properties. Herein, the surface functionalization of MgAlEu-layered double hydroxide (MAE-LDH) nanosheets on porous HAp scaffolds is reported as a simple and effective strategy to prepare HAp/MAE-LDH scaffolds for enhanced bone regeneration. The surface functionalization of MAE-LDHs on the porous HAp scaffold can significantly improve its surface roughness, specific surface, and hydrophilicity, thus effectively boosting the cells adhesion and osteogenic differentiation. Importantly, the MAE-LDHs grown on HAp scaffolds enable the sustained release of Mg2+ and Eu3+ ions for efficient bone repair and vascular regeneration. In vitro experiments suggest that the HAp/MAE-LDH scaffold presents much enhanced osteogenesis and angiogenesis properties in comparison with the pristine HAp scaffold. In vivo assays further reveal that the new bone mass and mineral density of HAp/MAE-LDH scaffold increased by 3.18- and 2.21-fold, respectively, than that of pristine HAp scaffold. The transcriptome sequencing analysis reveals that the HAp/MAE-LDH scaffold can activate the Wnt/β-catenin signaling pathway to promote the osteogenic and angiogenic abilities.

Porous hydroxyapatite scaffold orchestrated with bioactive coatings for rapid bone repair

Current bioceramic scaffolds for critical-size bone defects are still facing various challenges such as the poor capability of self-resorption, vascularization and osteogenesis. Herein, a composite scaffold (HOD) is fabricated by integrating bioactive coatings of konjac glucomannan (KGM) and deferoxamine (DFO) into porous hydroxyapatite scaffold (HA), where KGM coating induces the self-resorption of HOD after implanting and DFO promoted the vascularization at the defected bone. Porous HA scaffolds with 200–400 μm of pore sizes were prepared and these bioactive coatings were successfully deposited on the scaffold, which was confirmed by SEM. MC3T3-E1 cells could be tightly attached to the pore wall of HOD and the obvious osteogenic differentiation was clearly displayed after 14 days of co-culture. Besides, HOD displayed the potential of promoting the vascularization of HUVECs. Importantly, the accelerated degradation of HOD was observed in a macrophage-associated acidic medium, which led to the self-resorption of HOD in vivo. Micro-CT images showed that HOD was gradually replaced by newly formed bone, achieving a balance between the new bone formation and the scaffold degradation. The rapid bone repairing of the femoral defects in rats was displayed for HOD in comparison to the HA scaffold. Moreover, the therapeutic mechanism of HOD was highly associated with promoted osteogenesis and vascularization. Collectively, the porous ceramic scaffold orchestrated with bioactive coatings may be a promising strategy for repairing of the large bone defect.

A grooved porous hydroxyapatite scaffold induces osteogenic differentiation via regulation of PKA activity by upregulating miR-129-5p expression.

Hydroxyapatite scaffolds with different morphologies have been widely used in bone tissue engineering. Moreover, microRNAs (miRNAs) have been proven to be extensively involved in regulating bone regeneration. We developed grooved porous hydroxyapatite (HAG) scaffolds with good osteogenic efficiency. However, little is known about the role of miRNAs in HAG scaffold-mediated promotion of bone regeneration. The objective of this study was to reveal the mechanism from the perspective of differential miRNA expression. Scanning electron microscopy (SEM) was used to perform the coculture of cells and scaffolds. The miRNA profiles were generated by a microarray assay. A synthetic miR-129-5p mimic and inhibitor were used for overexpression or inhibition. The expression of osteogenic marker mRNAs and proteins was detected by quantitative real-time PCR (qRT-PCR), Western blotting, and immunofluorescence. An ALP activity kit and alizarin red staining (ARS) were used to measure ALP activity and mineral deposition formation. Cell migration ability was examined by wound healing and transwell assays. Protein kinase A (PKA) activity was measured by enzyme-linked immunosorbent assay (ELISA) after miR-129-5p transfection. Target genes were identified by a dual-luciferase reporter assay. H89 preculture evaluated the cross talk between miR-129-5p and PKA activity. Heterotopic implantation models, hematoxylin-eosin (HE), immunohistochemistry staining, and micro-CT were used to evaluate miR-129-5p osteogenesis in vivo. miRNAs were differentially expressed during osteogenic differentiation induced by HAG in vitro and in vivo. miR-129-5p was the only highly expressed miRNA both in vitro and in vivo. miR-129-5p overexpression promoted osteoblast differentiation and cell migration, while its inhibition weakened the effect of HAG. Moreover, miR-129-5p activated PKA to regulate the phosphorylation of β-catenin and cAMP-response element binding protein (CREB) by inhibiting cAMP-dependent protein kinase inhibitor alpha (Pkia). H89 prevented the effects of miR-129-5p on osteogenic differentiation and cell migration. HE, immunohistochemistry staining and micro-CT results showed that miR-129-5p promoted in vivo osteogenesis of the HAG scaffold. The HAG scaffold activates Pka by upregulating miR-129-5p and inhibiting Pkia, resulting in CREB-dependent transcriptional activation and accumulation of β-catenin and promoting osteogenic marker expression.