Biomimetic Periosteum Research Articles

A Bottom-Up Approach to Assemble Cell-Laden Biomineralized Nanofiber Mats into 3D Multilayer Periosteum Mimics for Bone Regeneration.

The creation of complex multilayer periosteal graft structures is challenging. This study introduced a novel bottom-up approach to assemble cell-laden nanofiber mats into a three-dimensional (3D) multilayer periosteum mimic, successfully replicating the hierarchical complexity of the natural periosteum. These nanofiber mats, which were fabricated by electrospinning, surface modification, and stimulated body fluid (SBF) immersion, are composed of nanoscale polycaprolactone (PCL) fibers coated with a mineralized collagen layer along the fiber orientation. They closely resembled the natural periosteal matrix, thereby promoting osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. The biomimetic periosteum, constructed via layer-by-layer assembly, offered advantages such as a multilayer nanofibrous structure, controlled cell distribution, a reservoir for osteoprogenitors, and enhanced pro-osteogenic potential. The rat calvarial bone defect model confirmed its potent bone repair capacity. This study presents an efficient approach to construct tissue-engineered periosteum mimics, holding promise for serving as periosteal grafts in orthopedic applications.

Construction of bilayer biomimetic periosteum based on SLA-3D printing for bone regeneration

An ideal biomimetic periosteum should have excellent biocompatibility to promote osteoclast adhesion and improve osseointegration, which is significant in promoting bone regeneration. In this work, a bionic artificial periosteum printed by the SLA-3D printing was prepared, consisting of a poly (ethylene glycol) diacrylate (PEGDA)/chitosan/tricalcium phosphate (TCP) fibrous layer and a gelatin methacryloyl (GelMA)/ammonium molybdate (Mo) cambium layer. Distinct surface characteristics were achieved on both sides of the biomimetic periosteum. Among them, the fibrous layer has high mechanical properties and low porosity, which is conducive to preventing the pulling of muscle tissues and the invasion of soft tissues. The cambium layer has a porous structure and bioactive factors that can effectively promote osteogenic differentiation of preosteoblasts. Combined with mild photothermal therapy triggered by NIR light, the biomimetic periosteum could promote bone regeneration at both the chemical and physical levels. This 3D-printed bilayer hydrogel can provide a promising strategy for preparing advanced tissue-engineered periosteum with excellent physical and bone regeneration properties.

Synergistic effect of ultrasound and reinforced electrical environment by bioinspired periosteum for enhanced osteogenesis via immunomodulation of macrophage polarization through Piezo1

The periosteum plays a vital role in repairing bone defects. Researchers have demonstrated the existence of electrical potential in the periosteum and native bone, indicating that electrical signals are essential for functional bone regeneration. However, the clinical use of external electrical treatments has been limited due to their inconvenience and inefficacy. As an alternative, low-intensity pulsed ultrasound (LIPUS) is a noninvasive form of physical therapy that enhances bone regeneration. Furthermore, the wireless activation of piezoelectric biomaterials through ultrasound stimulation would generate electric charges precisely at the defect area, compensating for the insufficiency of external electrical stimulation and potentially promoting bone regeneration through the synergistic effect of mechanical and electrical stimulation. However, the optimal integration of LIPUS with an appropriate piezoelectric periosteum is yet to be explored. Herein, the BaTiO3/multiwalled-carbon nanotubes/collagen (BMC) membranes have been fabricated, possessing physicochemical properties including improved surface hydrophilicity, enhanced mechanical performance, ideal piezoelectricity, and outstanding biocompatibility, all of which are conducive to bone regeneration. When combined with LIPUS, the endogenous electrical microenvironment of native bone was recreated. After that, the wireless-generated electrical signals, along with the mechanical signals induced by LIPUS, were transferred to macrophages and activated Ca2+ influx through Piezo1. Ultimately, the regenerative effect of the BMC membrane with LIPUS stimulation (BMC + L) was confirmed in a mouse cranial defect model. Together, this research presents a co-engineering strategy that involves fabricating a novel biomimetic periosteum and utilizing the synergistic effect of ultrasound to enhance bone regeneration, which is achieved through the reinforcement of the electrical environment and the immunomodulation of macrophage polarization.

Electrospun Biomimetic Periosteum Capable of Controlled Release of Multiple Agents for Programmed Promoting Bone Regeneration.

The effective repair of large bone defects remains a major challenge due to its limited self-healing capacity. Inspired by the structure and function of the natural periosteum, an electrospun biomimetic periosteum is constructed to programmatically promote bone regeneration using natural bone healing mechanisms. The biomimetic periosteum is composed of a bilayer with an asymmetric structure in which an aligned electrospun poly(ε-caprolactone)/gelatin/deferoxamine (PCL/GEL/DFO) layer mimics the outer fibrous layer of the periosteum, while a random coaxial electrospun PCL/GEL/aspirin (ASP) shell and PCL/silicon nanoparticles (SiNPs) core layer mimics the inner cambial layer. The bilayer controls the release of ASP, DFO, and SiNPs to precisely regulate the inflammatory, angiogenic, and osteogenic phases of bone repair. The random coaxial inner layer can effectively antioxidize, promoting cell recruitment, proliferation, differentiation, and mineralization, while the aligned outer layer can promote angiogenesis and prevent fibroblast infiltration. In particular, different stages of bone repair are modulated in a rat skull defect model to achieve faster and better bone regeneration. The proposed biomimetic periosteum is expected to be a promising candidate for bone defect healing.

A Self-Adaptive Biomimetic Periosteum Employing Nitric Oxide Release for Augmenting Angiogenesis in Bone Defect Regeneration.

The periosteum plays a vital role in the regeneration of critical-size bone defects and highly comminuted fractures, promoting the differentiation of osteoblasts, accelerating the reconstruction of the vascular network, and guiding bone tissue regeneration. However, the materials loaded with exogenous growth factors are limited by the release and activity of the elements. Therefore, the material structure must be carefully designed for the periosteal function. Here, a self-adaptive biomimetic periosteum strategy is proposed, which is a novel interpenetrating double network hydrogel consisting of diselenide-containing gelatin and calcium alginate (modified natural collagen and polysaccharide) to enhance the stability, anti-swelling, and delayed degradation of the hydrogel. The diselenide bond continuously releases nitric oxide (NO) by metabolizing endogenous nitrosated thiols (RSNO), activates the nitric oxide-cycle guanosine monophosphate (NO-cGMP) signal pathway, coordinates the coupling effect of angiogenesis and osteogenesis, and accelerates the repair of bone defects. This self-adaptive biomimetic periosteum with the interpenetrating double network structure formed by the diselenide-containing gelatin and calcium alginate has been proven to be safe and effective in repairing critical-size bone defects and is expected to provide a promising strategy for solving clinical problems.

Irisin-loaded electrospun core-shell nanofibers as calvarial periosteum accelerate vascularized bone regeneration by activating the mitochondrial SIRT3 pathway.

The scarcity of native periosteum poses a significant clinical barrier in the repair of critical-sized bone defects. The challenge of enhancing regenerative potential in bone healing is further compounded by oxidative stress at the fracture site. However, the introduction of artificial periosteum has demonstrated its ability to promote bone regeneration through the provision of appropriate mechanical support and controlled release of pro-osteogenic factors. In this study, a poly (l-lactic acid) (PLLA)/hyaluronic acid (HA)-based nanofibrous membrane was fabricated using the coaxial electrospinning technique. The incorporation of irisin into the core-shell structure of PLLA/HA nanofibers (PLLA/HA@Irisin) achieved its sustained release. In vitro experiments demonstrated that the PLLA/HA@Irisin membranes exhibited favorable biocompatibility. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) was improved by PLLA/HA@Irisin, as evidenced by a significant increase in alkaline phosphatase activity and matrix mineralization. Mechanistically, PLLA/HA@Irisin significantly enhanced the mitochondrial function of BMMSCs via the activation of the sirtuin 3 antioxidant pathway. To assess the therapeutic effectiveness, PLLA/HA@Irisin membranes were implanted in situ into critical-sized calvarial defects in rats. The results at 4 and 8 weeks post-surgery indicated that the implantation of PLLA/HA@Irisin exhibited superior efficacy in promoting vascularized bone formation, as demonstrated by the enhancement of bone matrix synthesis and the development of new blood vessels. The results of our study indicate that the electrospun PLLA/HA@Irisin nanofibers possess characteristics of a biomimetic periosteum, showing potential for effectively treating critical-sized bone defects by improving the mitochondrial function and maintaining redox homeostasis of BMMSCs.

Effect and mechanism of a concentration-dependent inorganic ion biomimetic periosteum in a repairing bone defect

An inorganic ion functionalized biomimetic periosteum constitutes a new strategy for the design and application of new bone repair materials. However, the effect of cumulative concentration of inorganic ions (e.g. inorganic ion (Si4+)) on bone immune microenvironment in promoting bone repair is still unclear. Here, we used different mesoporous bioactive glass nanoparticles (MBGNs) contents and a methacrylic acid gelatin (GelMA) organic–inorganic double crosslinking techniques to construct low, medium, and high content inorganic ion biomimetic periosteum. As the inorganic ion concentration increased, the compression modulus of LM@G, MM@G, and HM@G (1, 3, and 5 wt% MBGNs, respectively) was 1.6, 3.2, and 2.1 times higher than that of the GelMA hydrogel and the Si4+ release was 35.4 ± 0.4, 59.5 ± 0.2, and 71.5 ± 1.2 ppm in vitro, respectively. The ERK1/2 and p65 inhibition in the HM@G was 51.9% and 40.2% higher than that in the LM@G; however, the stem cell recruitment was about 85.2% lower than that in the LM@G, and the vascular germination degree was 35.4% lower than that in the MM@G. Contradiction between immune regulation and bone repair was found. However, the anti-inflammation in the MM@G was 30.8%, 30.1% higher than that in the LM@G, and stem cell recruitment and vascular sprouting were 3.4 and 1.6 times higher, respectively than those in the HM@G. In summary, the anti-inflammatory effect of MM@G group is stronger than that of LM@G group, and the effect of stem cell recruitment is more significant than that of HM@G group. We not only explore the biological mechanism of Si4+ coordinating macrophages and stem cells to promote bone repair, but also provide research basis for designing inorganic ion based immunofunctional biomaterials.

4D Printing of Personalized‐Tunable Biomimetic Periosteum with Anisotropic Microstructure for Accelerated Vascularization and Bone Healing (Adv. Healthcare Mater. 22/2023)

4D Printing of Personalized‐Tunable Biomimetic Periosteum with Anisotropic Microstructure for Accelerated Vascularization and Bone Healing (Adv. Healthcare Mater. 22/2023)

Seamlessly Adhesive Bionic Periosteum Patches Via Filling Microcracks for Defective Bone Healing.

Current artificial designs of the periosteum focus on osteogenic or angiogenic properties, while ignoring the filling and integration with bone microcracks, which trigger a prolonged excessive inflammatory reaction and lead to failure of bone regeneration. In this study, seamless adhesive biomimetic periosteum patches (HABP/Sr-PLA) were prepared to fill microcracks in defective bone via interfacial self-assembly induced by Sr ions mediated metal-ligand interactions among pamidronate disodium-modified hyaluronic acid (HAPD), black phosphorus (BP), and hydrophilic polylactic acid (PLA). In vitro, HABP/Sr-PLA exhibited excellent self-healing properties, seamlessly filled bone microcracks, and significantly enhanced osteogenesis and angiogenesis. Furthermore, in a rat cranial defect model, HABP/Sr-PLA was demonstrated to significantly promote the formation of blood vessels and new bone under mild 808 nm photothermal stimulation (42.8 °C), and the highest protein expression of CD31 and OPN was five times higher than that of the control group and other groups. Therefore, the proposed seamless microcrack-filled bionic periosteum patch is a promising clinical strategy for promoting bone repair.

4D Printing of Personalized-Tunable Biomimetic Periosteum with Anisotropic Microstructure for Accelerated Vascularization and Bone Healing.

An ideal biomimetic periosteum is expected to wrap various bone surfaces to orchestrate an optimal microenvironment for bone regeneration, including facilitating local vascularization, recruiting osteoblasts, and mineralizing the extracellular matrix (ECM). To mimic the role of the natural periosteum in promoting bone repair, a 4D printing technique to inlay aligned cell sheets on shape-shifting hydrogel is used, containing biophysical signals and spatially adjustable physical properties, for the first time. The outer hydrogel layer endows the biomimetic periosteum with the ability to digitally coordinate its 3D geometry to match the specific macroscopic bone shape to maintain a bone healing microenvironment. The inner aligned human mesenchymal stem cells (hMSCs) layer not only promotes the migration and angiogenesis of co-cultured cells but also exhibits excellent osteogenic differentiation properties. In vivo experiments show that apart from morphing preset shapes as physical barriers, the aligned biomimetic periosteum can actively facilitate local angiogenesis and early-stage osteogenesis. Altogether, this present work provides a novel route to construct a personalized biomimetic periosteum with anisotropic microstructure by introducing a tunable shape to maintain the bone reconstruction microenvironment and this strategy can be extended to repair sophisticated bone defects.

Endogenous Electric Field‐Coupled PD@BP Biomimetic Periosteum Promotes Bone Regeneration through Sensory Nerve via Fanconi Anemia Signaling Pathway (Adv. Healthcare Mater. 12/2023)

Biomimetic Periosteum In article 2203027 by Yanlin Su, Kaifang Chen, Daping Quan, Xiaodong Guo, and co-workers, [email protected] electroactive biomimetic periosteum is created, which can regulate the concentration of axon initial segments in sensory nerves, and promote bone regeneration by releasing neuropeptides. This will enrich the theory of neurogenic bone regeneration and provide a promising treatment for bone defects.

Functionalized acellular periosteum guides stem cell homing to promote bone defect repair

The periosteum plays a key role in bone tissue regeneration, especially in the promotion and protection of new bones. However, among the bone repair materials, many biomimetic artificial periosteum lack the natural periosteal structure, stem cells, and immunoregulation required for bone regeneration. In this study, we used natural periosteum to produce acellular periosteum. To retain the appropriate cell survival structure and immunomodulatory proteins, we grafted the functional polypeptide SKP on the surface collagen of the periosteum via an amide bond, providing the acellular periosteum with the ability to recruit mesenchymal stem cells. Thus, we developed a biomimetic periosteum (DP-SKP) with the ability to promote stem cell homing and immunoregulation in vivo. Compared to the blank and simple decellularized periosteum groups, DP-SKP was more conducive to stem cell adhesion, growth, and osteogenic differentiation in vitro. Additionally, compared with the other two groups, DP-SKP significantly promoted mesenchymal stem cell homing to the periosteal transplantation site, improved the bone immune microenvironment, and accelerated new lamellar bone formation in the critical size defect of rabbit skulls in vivo. Therefore, this acellular periosteum with a mesenchymal stem cell homing effect is expected to be used as an extracellular artificial periosteum in clinical practice.

Bioinspired Piezoelectric Periosteum to Augment Bone Regeneration via Synergistic Immunomodulation and Osteogenesis.

Ideal periosteum materials are required to participate in a sequence of bone repair-related physiological events, including the initial immune response, endogenous stem cell recruitment, angiogenesis, and osteogenesis. However, conventional tissue-engineered periosteal materials have difficulty achieving these functions by simply mimicking the periosteum via structural design or by loading exogenous stem cells, cytokines, or growth factors. Herein, we present a novel biomimetic periosteum preparation strategy to comprehensively enhance the bone regeneration effect using functionalized piezoelectric materials. The resulting biomimetic periosteum possessing an excellent piezoelectric effect and improved physicochemical properties was prepared using a biocompatible and biodegradable poly(3-hydroxybutyric acid-co-3-hydrovaleric acid) (PHBV) polymer matrix, antioxidized polydopamine-modified hydroxyapatite (PHA), and barium titanate (PBT), which were further incorporated into the polymer matrix to fabricate a multifunctional piezoelectric periosteum by a simple one-step spin-coating method. The addition of PHA and PBT dramatically enhanced the physicochemical properties and biological functions of the piezoelectric periosteum, resulting in improved surface hydrophilicity and roughness, enhanced mechanical performance, tunable degradation behavior, and stable and desired endogenous electrical stimulations, which is conducive to accelerating bone regeneration. Benefiting from endogenous piezoelectric stimulation and bioactive components, the as-fabricated biomimetic periosteum demonstrated favorable biocompatibility, osteogenic activity, and immunomodulatory functions in vitro, which not only promoted adhesion, proliferation, and spreading as well as osteogenesis of mesenchymal stem cells (MSCs) but also effectively induced M2 macrophage polarization, thereby suppressing reactive oxygen species (ROS)-induced inflammatory reactions. Through in vivo experiments, the biomimetic periosteum with endogenous piezoelectric stimulation synergistically accelerated the formation of new bone in a rat critical-sized cranial defect model. The whole defect was almost completely covered by new bone at 8 weeks post treatment, with a thickness close to that of the host bone. Collectively, with its favorable immunomodulatory and osteogenic properties, the biomimetic periosteum developed here represents a novel method to rapidly regenerate bone tissue using piezoelectric stimulation.

The high-strength and toughness Janus bionic periosteum matching bone development and growth in children

Pediatric periosteum requires suitable strength to stabilize bone injury and match the rapid growth of children's bone, as well as strong osteogenic and angiogenic capacity to promote bone repair. Here, a Janus membrane was constructed based on the natural gradient network of bacterial nano-cellulose (BNC), the molecular chain assembly of fish collagen (Col I) was achieved through the formation of amide bond from the amino group on the collagen and carboxyl group on the BNC surface after selective oxidization by using TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical). In situ synthesis of nano-hydroxyapatite (nHA) was conducted in the looser network of BNC by adjusting pH after addition in sequence of Ca(NO3)2·4H2O and Na3PO4 solution. The Janus membrane possessed Young's modulus and tensile capacity within the ligament range (0.113 GPa and 29.63 MPa, respectively), which allow material-bone fixation and integration. In vitro biomolecular experiments showed that the Janus periosteum promoted the proliferation of bone marrow mesenchymal stem cells and the expression of angiogenic and osteogenic factors. The promotions in angiogenesis and osteogenesis were further verified in vivo. Overall, the Janus biomimetic periosteum met the requirements of growth and development in children, indicating its regenerative potential for the treatment of periosteum-associated diseases in children.

Micropatterned photothermal double-layer periosteum with angiogenesis-neurogenesis coupling effect for bone regeneration.

The abundant neurovascular network in the periosteal fibrous layer is essential for regulating bone homeostasis and repairing bone defects. However, the majority of the current studies only focus on the structure or function, and most of them merely involve osteogenesis and angiogenesis, lacking an in-depth study of periosteal neurogenesis. In this study, a photothermal double-layer biomimetic periosteum with neurovascular coupling was proposed. The outer layer of biomimetic periosteum is a conventional electrospinning membrane to prevent soft tissue invasion, and the inner layer is an oriented nanofiber membrane to promote cell recruitment and angiogenesis. From the perspective of functional bionics, based on the whitlockite (WH) similar to bone composition, we doped Nd (the trivalent form of neodymium element) in it as the inducing element of photothermal response to prepare photothermal whitlockite (Nd@WH). The sustained release of Mg2+ in Nd@WH can effectively promote the up-regulation of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF). The release of Ca2+ and PO4 3- ions and photothermal osteogenesis jointly promote bone regeneration. Under the combined effect of structure and function, the formation of nerves, blood vessels, and related collagens greatly simulates the microenvironment of extracellular matrix and periosteum regeneration and ultimately promotes bone regeneration. In this study, physical and chemical characterization proved that the bionic periosteum has good flexibility and operability. The in vitro cell experiment and in vivo calvarial defect model verified that PPCL/Nd@WH biomimetic periosteum had excellent bone tissue regeneration function compared with other groups. Finally, PPCL/Nd@WH provides a new idea for the design of bionic periosteum.

Endogenous Electric Field-Coupled PD@BP Biomimetic Periosteum Promotes Bone Regeneration through Sensory Nerve via Fanconi Anemia Signaling Pathway.

To treat bone defects, repairing the nerve-rich periosteum is critical for repairing the local electric field. In this study, an endogenous electric field is coupled with 2D black phosphorus electroactive periosteum to explore its role in promoting bone regeneration through nerves. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are used to characterize the electrically active biomimetic periosteum. Here, the in vitro effects exerted by the electrically active periosteum on the transformation of Schwann cells into the repair phenotype, axon initial segment (AIS) and dense core vesicle (DCV) of sensory neurons, and bone marrow mesenchymal stem cells are assessed using SEM, immunofluorescence, RNA-sequencing, and calcium ion probes. The electrically active periosteum stimulates Schwann cells into a neuroprotective phenotype via the Fanconi anemia pathway, enhances the AIS effect of sensory neurons, regulates DCV transport, and releases neurotransmitters, promoting the osteogenic transformation of bone marrow mesenchymal stem cells. Microcomputed tomography and other in vivo techniques are used to study the effects of the electrically active periosteum on bone regeneration. The results show that the electrically active periosteum promotes nerve-induced osteogenic repair, providing a potential clinical strategy for bone regeneration.

Poly (l-lactic acid)-based modified nanofibrous membrane with dual drug release capability for GBR application

Electrospun multilayer nanofibers guided bone regeneration (GBR) with a new design were developed in this study. The synthesized multilayer GBR was composed of two distinct layers. Poly l-lactic acid (PLA) incorporated with simvastatin (SIM) was designed as PLA/SIM layer to contact with a bone defect. In addition, the hydrophilic gelatin (GT) containing thymol (THY) was fabricated as GT/THY layer to contact connective tissue, potentially for bacterial gathering. Due to the different chemical nature and weak cohesion of the hydrophilic and hydrophobic layers, hybrid fibers made of PLA/SIM and GT/THY were electrospun as cohesion promoters between these layers. The microstructure and characteristics of the synthesized multilayer substrate, named GT/PLA, were evaluated, and different fibrous monolayers were fabricated to determine the optimal concentrations of drugs.Scanning electron microscopy (SEM) images showed continuous, smooth, randomly aligned, and bead-free fibers. In addition, there were no drug particles on the fiber surfaces which displayed the good placement of those inside the fibers. The mats exhibited satisfactory tensile strength (4.60 ± 0.14 MPa) and favorable physicochemical properties, including proper porosity percentage (<50 %) and appropriate pore size. Suitable swelling behavior (293 ± 0.05 %) and adequate degradation rates were also approved by characterizing swelling and degradability in vitro. The GT/PLA membrane exhibited a prolonged and sustained SIM release and controlled THY release with high antibacterial efficiency. Cell viability, cell attachment assay, and nuclear staining using 4′,6-diamidino-2-phenylindole (DAPI) showed that the designed GT/PLA substrate had good biocompatibility and cell attachment. Cell infiltration testing also showed that the cells were finely prevented by the outer layer (GT/THY). Overall, the obtained results in this study indicated the great potential of the prepared GT/PLA for use as a GBR which can develop osteogenic and antibacterial biomimetic periosteum optimizing the clinical application of GBR strategies.

Engineered periosteum-diaphysis substitutes with biomimetic structure and composition promote the repair of large segmental bone defects

The repair of large segmental bone defects remains a big challenge due to limited self-healing capacity of bone. Digging into the structure and composition of natural long bone let us realize that the periosteum cambium on the surface of diaphysis plays a crucial role in bone repair. In this study, we explored the feasibility of using a tissue-engineered periosteum-diaphysis substitute to repair the large segmental bone defects. To create an artificial periosteum cambium, bone marrow mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs) were co-cultured on electrospun silk fibroin (SF) fibrous membranes for mimicking the cellular composition and microstructure of cambium layer of the native periosteum. These SF membranes supported the adhesion and proliferation of both BMSCs and EPCs. In addition, we found that a 1:1 ratio of BMSCs and EPCs supported osteogenesis and angiogenesis optimally. This biomimetic periosteum layer was integrated with artificial diaphysis made of tubular SF scaffolds to construct a biomimetic periosteum-diaphysis substitute. Animal studies confirmed that the biomimetic periosteum-diaphysis substitutes promoted the repair of critical-size bone defects of rabbit radius. Furthermore, the transplanted biomimetic periosteum-diaphysis substitute could prevent the growth of fibrous tissues in the bone defect, and thus reduce the occurrence of nonunion. This study described a promising tissue engineering strategy for the repair of large segmental bone defects.

A multifaceted biomimetic periosteum with a lamellar architecture and osteogenic/angiogenic dual bioactivities.

An artificial periosteum has emerged as an encouraging candidate for bone defect repair. Currently, it remains a great challenge to develop a multifaceted biomimetic periosteum integrating multifunctional features of bioactivities and unique mechanical properties. Here, we successfully fabricated an artificial periosteum (AP) composed of hierarchically assembled Mg-doped mineralized collagen microfibrils with a biomimetically rotated lamellar structure via a "multiscale cascade regulation" strategy combining multiple techniques such as molecular self-assembly, electrospinning, and pressure-driven fusion from molecular to macroscopic levels. The AP has excellent mechanical properties with an ultimate strength and a tensile modulus of 15.9 MPa and 1.1 GPa, respectively. The involvement of Mg-doped nano-hydroxyapatite endowed the AP with good osteogenic and angiogenic activities to promote osteogenic differentiation of bone marrow mesenchymal stem cells and human umbilical vein endothelial cell differentiation into capillary-like structures in vitro. In addition, the results of in vivo evaluations in a rat cranial bone defect model including micro-CT morphology, histological staining, and immunohistochemical analysis showed that Mg-doped mineralized collagen-based AP (MgMC@AP) significantly facilitated cranial bone regeneration and fast vascularization. Our findings suggest that the AP mimicked the composition, lamellar structure, mechanical properties, and biological activities of natural periosteum/lamellae, showing great promise for bone tissue regeneration.

Construction of biomimetic cell-sheet-engineered periosteum with a double cell sheet to repair calvarial defects of rats.

BackgroundThe periosteum plays a crucial role in the development and injury healing process of bone. The purpose of this study was to construct a biomimetic periosteum with a double cell sheet for bone tissue regeneration.MethodsIn vitro, the human amniotic mesenchymal stem cells (hAMSCs) sheet was first fabricated by adding 50 ​μg/ml ascorbic acid to the cell sheet induction medium. Characterization of the hAMSCs sheet was tested by general observation, microscopic observation, live/dead staining, scanning electron microscopy (SEM) and hematoxylin and eosin (HE) staining. Afterwards, the osteogenic cell sheet and vascular cell sheet were constructed and evaluated by general observation, alkaline phosphatase (ALP) staining, Alizarin Red S staining, SEM, live/dead staining and CD31 immunofluorescent staining for characterization. Then, we prepared the double cell sheet. In vivo, rat calvarial defect model was introduced to verify the regeneration of bone defects treated by different methods. Calvarial defects (diameter: 4 ​mm) were created of Sprague–Dawley rats. The rats were randomly divided into 4 groups: the control group, the osteogenic cell sheet group, the vascular cell sheet group and the double cell sheet group. Macroscopic, micro-CT and histological evaluations of the regenerated bone were performed to assess the treatment results at 8 weeks and 12 weeks after surgery.ResultsIn vitro, hAMSCs sheet was successfully prepared. The hAMSCs sheet consisted of a large number of live hAMSCs and abundant extracellular matrix (ECM) that secreted by hAMSCs, as evidenced by macroscopic/microscopic observation, live/dead staining, SEM and HE staining. Besides, the osteogenic cell sheet and the vascular cell sheet were successfully prepared, which were verified by general observation, ALP staining, Alizarin Red S staining, SEM and CD31 immunofluorescent staining. In vivo, the macroscopic observation and micro-CT results both demonstrated that the double cell sheet group had better effect on bone regeneration than other groups. In addition, histological assessments indicated that large amounts of new bone had formed in the calvarial defects and more mature collagen in the double cell sheet group.ConclusionThe double cell sheet could promote to repair calvarial defects of rats and accelerate bone regeneration.The translational potential of this articleWe successfully constructed a biomimetic cell-sheet-engineered periosteum with a double cell sheet by a simple, low-cost and effective method. This biomimetic periosteum may be a promising therapeutic strategy for the treatment of bone defects, which may be used in clinic in the future.