Tissue-engineered Periosteum Research Articles

Nano artificial periosteum PCL/Ta/ZnO accelerates repair of periosteum via antibacterial, promoting vascularization and osteogenesis

The periosteum plays a critical role in bone development, shaping, remodeling, and fracture healing due to its abundance of osteoprogenitor cells, osteoblasts, and capillary network. However, the role of periosteum in bone injury healing has been underestimated, thus there is an urgent need to develop a multifunctional artificial periosteum that mimics the natural one. To tackle this issue, electrospinning technology was employed to fabricate an artificial periosteum composed of Poly-ε-caprolactone (PCL) doped with tantalum (Ta) and zinc oxide (ZnO) nanoparticles to enhance its antibacterial, osteogenic, and angiogenic properties. The in vitro cell experiments have demonstrated that the PCL/Ta/ZnO artificial periosteum exhibits excellent biocompatibility and can effectively facilitate osteogenic differentiation of BMSCs as well as angiogenic differentiation of EPCs. Antibacterial experiments have demonstrated the excellent bactericidal effects of PCL/Ta/ZnO artificial periosteum against both S. aureus and E. coli. The subcutaneous infection and critical-sized skull bone defect models have validated its in vivo properties of antibacterial activity, promotion of osteogenesis, and angiogenic potential. The PCL/Ta/ZnO artificial periosteum demonstrates remarkable efficacy in infection control and favorable immunomodulation, thereby achieving rapid vascularized bone repair. In conclusion, the utilization of PCL/Ta/ZnO tissue-engineered periosteum has been demonstrated to exhibit antibacterial properties, pro-vascularization effects, and promotion of osteogenesis at the site of bone defects. This promising approach could potentially offer effective treatment for bone defects.

Selective Formation of Osteogenic and Vasculogenic Tissues for Cartilage Regeneration.

Tissue-engineered periosteum substitutes (TEPSs) incorporating hierarchical architecture with osteoprogenitor and vascular niches are drawing much attention as a promising tool to support functional cells in defined zones and nourish the cortical bone. Current TEPSs usually lack technologies to closely observe cell performance, especially at the cell contact interface between distinct compartments containing defined biological configurations and functions. Here, an electrodeposition strategy is reported, which enables the selective formation of TEPSs with osteoprogenitor and vascular niches in a multiphasic scaffold in combination with different human cell types for cartilage regeneration in an in vivo osteochondral defect model. Human umbilical vein endothelial cells (HUVECs), dermal fibroblasts (HDFs), and bone marrow mesenchymal stem cells (hMSCs) are used to mirror both the vascular and osteogenic niches, respectively. It is observed that the intrinsic viscoelastic nature of the porous solid matrix is essential to successfully induce angiogenesis. Coculture of hMSCs with functional cells (HUVECs/HDFs) in TEPSs also effectively promoted periosteal regeneration, including osteogenic and angiogenic processes. The osteoarthritis cartilage histopathology assessment and histologic/histochemical grading system data indicate that the TEPSs containing hMSCs/HUVECs/HDFs exhibit superior potential for cartilage regeneration.

Small Intestinal Submucosa Biomimetic Periosteum Promotes Bone Regeneration.

Background: Critical bone defects are a significant problem in clinics. The periosteum plays a vital role in bone regeneration. A tissue-engineered periosteum (TEP) has received increasing attention as a novel strategy for bone defect repairs. Methods: In this experiment, a biomimetic periosteum was fabricated by using coaxial electrospinning technology with decellularized porcine small intestinal submucosa (SIS) as the shell and polycaprolactone (PCL) as the core. In vitro, the effects of the biomimetic periosteum on Schwann cells, vascular endothelial cells, and bone marrow mesenchymal stem cells were detected by a scratch test, an EdU, a tube-forming test, and an osteogenesis test. In vivo, we used HE staining to evaluate the effect of the biomimetic periosteum on bone regeneration. Results: In vitro experiments showed that the biomimetic periosteum could significantly promote the formation of angiogenesis, osteogenesis, and repaired Schwann cells (SCs). In vivo experiments showed that the biomimetic periosteum could promote the repair of bone defects. Conclusions: The biomimetic periosteum could simulate the structural function of the periosteum and promote bone repair. This strategy may provide a promising method for the clinical treatment of skull bone defects.

Development and fabrication of co-axially electrospun biomimetic periosteum with a decellularized periosteal ECM shell/PCL core structure to promote the repair of critical-sized bone defects

Periosteum is crucial to the initial healing of bone defects because it provides a stable periosteum-specific microenvironment and abundant osteogenic cells towards bone repair. However, the usage of autologous periosteum is restricted for limited availability. Xenogeneic decellularized periosteum (DP) preserves periosteum-specific cues but the hardly-controlled physicochemical properties render it inconvenient to use widely. Our study aimed to fabricate a tissue-engineered periosteum (TEP) with a continuous periosteum-specifically bioactive surface and tailored physicochemical properties by co-axial electrospinning of poly(ε-caprolactone) (PCL)/periosteal decellularized extracellular matrix (dECM) to promote bone defects healing. Before use, DP was confirmed for effective removal of residual DNA and well preservation of biological components, e.g., collagen and glycosaminoglycans . After optimization of fabrication processes , PCL/dECM co-axially electrospun membrane (PEC) bore an exquisite core-shell structure, which was effectively equipped with composite advantages of dECM and PCL. Notably, PEC stood out in terms of tensile strength and long-term durability within a physiological environment compared to that of the dECM uniaxial electrospun membrane (ECM). Besides, PEC exhibited remarkably better cell proliferation , migration, bio-mineralization, and osteogenic properties as compared to that of the electrospun PCL membrane. Moreover, PEC-TEP could significantly enhance the recovery of critical-sized bone defects in rats than that of PCL-TEP. To the best of our knowledge, biomimetic PEC-TEP was fabricated and used for the first time to repair bone defects. This novel strategy is promising to fabricate a dECM-based advanced membrane for tissue engineering.

Periosteum and development of the tissue-engineered periosteum for guided bone regeneration.

BackgroundPeriosteum plays a significant role in bone formation and regeneration by storing progenitor cells, and also acts as a source of local growth factors and a scaffold for recruiting cells and other growth factors. Recently, tissue-engineered periosteum has been studied extensively and shown to be important for osteogenesis and chondrogenesis. Using biomimetic methods for artificial periosteum synthesis, membranous tissues with similar function and structure to native periosteum are produced that significantly improve the efficacy of bone grafting and scaffold engineering, and can serve as direct replacements for native periosteum. Many problems involving bone defects can be solved by preparation of idealized periosteum from materials with different properties using various techniques.MethodsThis review summarizes the significance of periosteum for osteogenesis and chondrogenesis from the aspects of periosteum tissue structure, osteogenesis performance, clinical application, and development of periosteum tissue engineering. The advantages and disadvantages of different tissue engineering methods are also summarized.ResultsThe fast-developing field of periosteum tissue engineering is aimed toward synthesis of bionic periosteum that can ensure or accelerate the repair of bone defects. Artificial periosteum materials can be similar to natural periosteum in both structure and function, and have good therapeutic potential. Induction of periosteum tissue regeneration and bone regeneration by biomimetic periosteum is the ideal process for bone repair.ConclusionsPeriosteum is essential for bone formation and regeneration, and it is indispensable in bone repair. Achieving personalized structure and composition in the construction of tissue engineering periosteum is in accordance with the design concept of both universality and emphasis on individual differences and ensures the combination of commonness and individuality, which are expected to meet the clinical needs of bone repair more effectively.The translational potential of this articleTo better understand the role of periosteum in bone repair, clarify the present research situation of periosteum and tissue engineering periosteum, and determine the development and optimization direction of tissue engineering periosteum in the future. It is hoped that periosteum tissue engineering will play a greater role in meeting the clinical needs of bone repair in the future, and makes it possible to achieve optimization of bone tissue therapy.

Repair of long bone defects of large size using a tissue-engineered periosteum in a rabbit model

Tissue engineering is a promising approach for bone regeneration. In this study, we aimed to investigate whether tissue engineered periosteum (TEP), which was fabricated by combining osteogenically-induced mesenchymal stem cells (MSCs) with porcine small intestinal submucosa (SIS), could restore long bone defects of large size in rabbits. Twenty-four adult New Zealand white rabbits (NZWRs) were used in the experiments. Long bone defects of large size (30 mm-50 mm; average, 40 mm) were established on both sides of NZWRs’ radii. The defects were treated with TEP (Group A), allogeneic deproteinized bone (DPB, Group B), TEP combined with DPB (Group C), and pure SIS (Group D). The healing outcome was evaluated by radiography and histological examination at 4, 8, and 12 weeks post-treatment. The radiographical findings showed that bone defects of large size were all repaired in Groups A, B and C within 12 weeks, whereas Group D (pure SIS group) failed to result in defect healing at 4, 8, and 12 weeks. Although there was some new bone regeneration connecting the allografts and bone ends, as observed under radiographical and histological observations, bone defects of large sizes were restored primarily by structurally allografted DPB within 12 weeks. The TEP groups (Groups A and C) showed partial or total bone regeneration upon histological inspection. Based on 12-week histological examinations, significantly more bone was formed in Group A than Group C (P < 0.05), and both groups formed significantly more bone than in Groups B and D. The results indicated that long bone defects of a large size could be restored by TEP or TEP combined with the DPB scaffold, and such materials provide an alternative approach to resolving pathological bone defects in clinical settings.

Periosteal Tissue Engineering: Current Developments and Perspectives.

Periosteum, a highly vascularized bilayer connective tissue membrane plays an indispensable role in the repair and regeneration of bone defects. It is involved in blood supply and delivery of progenitor cells and bioactive molecules in the defect area. However, sources of natural periosteum are limited, therefore, there is a need to develop tissue-engineered periosteum (TEP) mimicking the composition, structure, and function of natural periosteum. This review explores TEP construction strategies from the following perspectives: i) different materials for constructing TEP scaffolds; ii) mechanical properties and surface topography in TEP; iii) cell-based strategies for TEP construction; and iv) TEP combined with growth factors. In addition, current challenges and future perspectives for development of TEP are discussed.

Matrix metalloproteinase (MMP)-degradable tissue engineered periosteum coordinates allograft healing via early stage recruitment and support of host neurovasculature

Despite serving as the clinical “gold standard” treatment for critical size bone defects, decellularized allografts suffer from long-term failure rates of ~60% due to the absence of the periosteum. Stem and osteoprogenitor cells within the periosteum orchestrate autograft healing through host cell recruitment, which initiates the regenerative process. To emulate periosteum-mediated healing, tissue engineering approaches have been utilized with mixed outcomes. While vascularization has been widely established as critical for bone regeneration, innervation was recently identified to be spatiotemporally regulated together with vascularization and similarly indispensable to bone healing. Notwithstanding, there are no known approaches that have focused on periosteal matrix cues to coordinate host vessel and/or axon recruitment. Here, we investigated the influence of hydrogel degradation mechanism, i.e. hydrolytic or enzymatic (cell-dictated), on tissue engineered periosteum (TEP)-modified allograft healing, especially host vessel/nerve recruitment and integration. Matrix metalloproteinase (MMP)-degradable hydrogels supported endothelial cell migration from encapsulated spheroids whereas no migration was observed in hydrolytically degradable hydrogels in vitro, which correlated with increased neurovascularization in vivo. Specifically, ~2.45 and 1.84-fold, and ~3.48 and 2.58-fold greater vessel and nerve densities with high levels of vessel and nerve co-localization was observed using MMP degradable TEP (MMP-TEP) -modified allografts versus unmodified and hydrolytically degradable TEP (Hydro-TEP)-modified allografts, respectively, at 3 weeks post-surgery. MMP-TEP-modified allografts exhibited greater longitudinal graft-localized vascularization and endochondral ossification, along with 4-fold and 2-fold greater maximum torques versus unmodified and Hydro-TEP-modified allografts after 9 weeks, respectively, which was comparable to that of autografts. In summary, our results demonstrated that the MMP-TEP coordinated allograft healing via early stage recruitment and support of host neurovasculature.

Irregular Bone Defect Repair Using Tissue-Engineered Periosteum in a Rabbit Model

Background:In previous studies, we succeeded in repairing a long bone defect with tissue-engineered periosteum (TEP), fabricated by incorporating rabbit mesenchymal stem cells with small intestinal submucosa. In this study, we investigated the feasibility of allogeneic irregular bone defect repair using TEP.Methods:We performed a subtotal resection of the scapula in 36 rabbits to establish a large irregular bone defect model. The rabbits were then randomly divided into three groups (n = 12 per group) and the defects were treated with TEP (Group 1), allogeneic deproteinized bone (DPB) (Group 2) or a hybrid of TEP and DPB (Group 3). At 4, 8, and 12 weeks after surgery, the rabbits were sacrificed, and the implants were harvested. X-ray radiographic and histological examinations were performed to detect bone healing. Ink-formaldehyde perfusion was introduced to qualitatively analyze vascularization in TEP engineered new bone.Results:The repair of scapular defects was diverse in all groups, shown by radiographic and histological tests. The radiographic scores in Group 1 and Group 3 were significantly higher than Group 2 at 8 and 12 weeks (p < 0.05). Histological scores further proved that Group 1 had significantly greater new bone formation compared to Group 3 (p < 0.05), while Group 2 had the lowest osteogenesis at all time-points (p < 0.001). Ink-formaldehyde perfusion revealed aboundant microvessels in TEP engineered new bone.Conclusion:We conclude that TEP is promising for the repair of large irregular bone defects. As a 3D scaffold, DPB could provide mechanical support and a shaping guide when combined with TEP. TEP engineered new bone has aboundant microvessels.

Advances in the Masquelet technique: Myeloid-derived suppressor cells promote angiogenesis in PMMA-induced membranes

The periosteum plays a critical role in bone formation and defect reconstruction. The concept of tissue engineering in the periosteum has been suggested to solve the clinical problems related to bone defect repair. Insertion of polymethyl methacrylate (PMMA) bone cement can induce the autologous generation of a tissue-engineered periosteum and has been considered as a promising strategy for bone defect reconstruction. The PMMA-induced membrane is a crucial element in the reconstruction of bone defects, especially for angiogenesis, but its biological mechanism remains elusive. Here, a PMMA-induced membrane model was established using a femoral critically sized defect in mice. We identified myeloid-derived suppressor cells (MDSCs) as a regulatory component of induced membrane vascularization. The increased number of MDSCs was markedly linked to increased membrane thickness and capillary density. Importantly, the results of an in vitro coculture assay indicated that MDSCs of the induced membrane further facilitated the angiogenic capacity of human umbilical vein endothelial cells (HUVECs) by upregulating the expression of VEGFA, Ang2 and HIF-1α. Furthermore, signaling pathway blockade results suggested that STAT3 activation is involved in the upregulation of VEGFA, Ang2 and HIF-1α expression in induced membrane MDSCs. Our findings provide new insights into the mechanism of angiogenesis in the PMMA-induced membrane and confirm the key signaling molecules of MDSCs in induced membrane angiogenesis. Based on these results, this strategy may become a new therapy for the treatment of large bone defects in the future. Statement of SignificanceIn this study, we established an autologous tissue-engineered periosteum – PMMA-induced membrane, which was formed by the foreign body reaction to PMMA bone cement. The induced membrane establishes a blood supply for the large bone defect healing. After investigation, our study discovered the critical cell type in the formation and angiogenesis processes of the induced membrane, myeloid-derived suppressor cells (MDSCs). We revealed that MDSCs of the induced membrane promote the angiogenesis of endothelial cells through the expression of VEGFA, Ang2 and HIF-1α, which was upregulated by the activation of STAT3 signaling. Our findings clarified the beneficial effect of MDSCs in the angiogenesis of bone repair, and offered an additional target for the study of foreign body reactions to bone repair materials.

A sulfonated PEEK/PCL composite nanofibrous membrane for periosteum tissue engineering application

The favorable mechanical property and regenerative capacity of bone graft are extremely important for the reconstruction of the large bone defect. Polyetheretherketone (PEEK) has tailored elastic modulus to natural cortical bone, while its application is limited by bio-inert surface. Tissue-engineered periosteum exhibits improved osteogenesis-inducing capacity. However, the present artificial periosteum is difficult to effectively anchor to bone grafts. The use of PEEK-based nanomembrane may be a promising candidate to integrate with PEEK bone graft without anchor problem, which has not been reported until now. In this work, a series of fluorinated PEEK polymers were successfully synthesized and electrospun into nanofibers. The chemical structure, morphology, thermal stability, wettability and mechanical strength of s-PEEK membranes were investigated. Then, a novel flexible nanocomposite membrane for tissue-engineered periosteum based on fibrous sulfonated PEEK (s-PEEK) and polycaprolactone (PCL) was prepared. The functional properties were evaluated accordingly. The s-PEEK/PCL composite membranes showed superior hydrophilic and better ductility than pure s-PEEK film. The capacity of protein adsorption was also evaluated, suggesting the elevated bioactivity of s-PEEK/PCL composite membranes. Finally, the composite membranes were successfully mineralized by a homogenous and thinner calcium phosphate layer, indicating its potential to improve the osteogenic response.

Layer-by-layer nanofiber-enabled engineering of biomimetic periosteum for bone repair and reconstruction

Periosteum plays an indispensable role in bone repair and reconstruction. To recapitulate the remarkable regenerative capacity of periosteum, a biomimetic tissue-engineered periosteum (TEP) was constructed via layer-by-layer bottom-up strategy utilizing polycaprolactone (PCL), collagen, and nano-hydroxyapatite composite nanofiber sheets seeded with bone marrow stromal cells (BMSCs). When combined with a structural bone allograft to repair a 4 mm segmental bone defect created in the mouse femur, TEP restored donor-site periosteal bone formation, reversing the poor biomechanics of bone allograft healing at 6 weeks post-implantation. Further histologic analyses showed that TEP recapitulated the entire periosteal bone repair process, as evidenced by donor-dependent formation of bone and cartilage, induction of distinct CD31high type H endothelium, reconstitution of bone marrow and remodeling of bone allografts. Compared to nanofiber sheets without BMSC seeding, TEP eliminated the fibrotic tissue capsule elicited by nanofiber sheets, leading to a marked improvement of osseointegration at the compromised periosteal site. Taken together, our study demonstrated a novel layer-by-layer engineering platform for construction of a versatile biomimetic periosteum, enabling further assembly of a multi-component and multifunctional periosteum replacement for bone defect repair and reconstruction.

Controlled release of FGF-2 and BMP-2 in tissue engineered periosteum promotes bone repair in rats

The aim of this study was to prepare chitosan–collagen (CS/COL) scaffolds that could release fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein 2 (BMP-2), and to study the effect of this scaffold on bone repair. By improving the double emulsion/solvent evaporation technique, BMP-2 was encapsulated in poly(lactic acid)–poly(ethylene glycol)–poly(lactic acid) (PELA) microcapsules, to the surface of which FGF-2 was attached. The CS/COL scaffold carrying the microcapsules was prepared by freeze-drying. Periosteum derived cells (PDCs) were extracted and cultured on the scaffolds to study their proliferation and differentiation on the scaffolds. In addition, the effects of the scaffolds were investigated on rats with skull defects by micro-computed tomography and histology. We successfully prepared PELA microcapsules with external adherence to FGF-2 and encapsulated with BMP-2. The CS/COL scaffolds were porous and PDCs adhered, proliferated and underwent osteogenic differentiation on the scaffolds. The sequential release of FGF-2/BMP-2 had better osteogenic efficacy than other groups. Our results suggest that CS/COL scaffolds that bind FGF-2 and BMP-2 in combination with PDCs could be a promising new strategy for tissue engineering periosteum.

Artificial periosteum in bone defect repair—A review

Periosteum is a thin membrane that encases the surfaces of most bones. It is composed of an outer fibrous layer contains longitudinally oriented cells and collagen fibers and an inner cambial layer that consists of multipotent mesenchymal stem cells (MSCs) and osteogenic progenitor cells. Periosteum has a function of regulating cell and collagen arrangement, which is important to the integrity, modelling and remodelling of bone, particularly during bone defect repair. Apart from autograft and allograft, artificial periosteum, or tissue-engineered periosteum mimicking native periosteum in structure or function, made up of small intestinal submucosa, acellular dermis, induced membrane, cell sheets, and polymeric scaffolds, and so on, has been developed to be used in bone defect repair. In this review, we classify the artificial periosteum into three approaches based on the material source, that is, native tissues, scaffold-free cell sheets and scaffold-cell composites. Mechanisms, methods and efficacy of each approach are provided. Existing obstacles and enabling technologies for future directions are also discussed.

In vivo investigation of tissue-engineered periosteum for the repair of allogeneic critical size bone defects in rabbits.

The aim of the study was to evaluate the efficacy of tissue-engineered periosteum (TEP) in repairing allogenic bone defects in the long term. TEP was biofabricated with osteoinduced rabbit bone marrow mesenchymal stem cells and porcine small intestinal submucosa (SIS). A total of 24 critical sized defects were created bilaterally in radii of 12 New Zealand White rabbits. TEP/SIS was implanted into the defect site. Bone defect repair was evaluated with radiographic and histological examination at 4, 8 and 12 weeks. Bone defects were structurally reconstructed in the TEP group with mature cortical bone and medullary canals, however this was not observed in the SIS group at 12 weeks. The TEP approach can effectively restore allogenic critical sized defects, and achieve maturity of long-bone structure in 12 weeks in rabbit models.

Periosteum tissue engineering in an orthotopic invivo platform.

The periosteum plays a critical role in bone homeostasis and regeneration. It contains a vascular component that provides vital blood supply to the cortical bone and an osteogenic niche that acts as a source of bone-forming cells. Periosteal grafts have shown promise in the regeneration of critical size defects, however their limited availability restricts their widespread clinical application. Only a small number of tissue-engineered periosteum constructs (TEPCs) have been reported in the literature. A current challenge in the development of appropriate TEPCs is a lack of pre-clinical models in which they can reliably be evaluated. In this study, we present a novel periosteum tissue engineering concept utilizing a multiphasic scaffold design in combination with different human cell types for periosteal regeneration in an orthotopic invivo platform. Human endothelial and bone marrow mesenchymal stem cells (BM-MSCs) were used to mirror both the vascular and osteogenic niche respectively. Immunohistochemistry showed that the BM-MSCs maintained their undifferentiated phenotype. The human endothelial cells developed into mature vessels and connected to host vasculature. The addition of an invitro engineered endothelial network increased vascularization in comparison to cell-free constructs. Altogether, the results showed that the human TEPC (hTEPC) successfully recapitulated the osteogenic and vascular niche of native periosteum, and that the presented orthotopic xenograft model provides a suitable invivo environment for evaluating scaffold-based tissue engineering concepts exploiting human cells.

Non-invasive diffuse correlation tomography reveals spatial and temporal blood flow differences in murine bone grafting approaches.

Longitudinal blood flow during murine bone graft healing was monitored non-invasively using diffuse correlation tomography. The system utilized spatially dense data from a scanning set-up, non-linear reconstruction, and micro-CT anatomical information. Weekly in vivo measurements were performed. Blood flow changes in autografts, which heal successfully, were localized to graft regions and consistent across mice. Poor healing allografts showed heterogeneous blood flow elevation and high inter-subject variabilities. Allografts with tissue-engineered periosteum showed responses intermediate to both autografts and allografts, consistent with healing observed. These findings suggest that spatiotemporal blood flow changes can be utilized to differentiate the degree of bone graft healing.

Hydroxyapatite-containing silk fibroin nanofibrous scaffolds for tissue-engineered periosteum

A simple, one-step technology is developed to generate a hydroxyapatite (HA)-containing silk fibroin nanofibrous scaffold which has great potential as osteogenesis promoting scaffolds for constructing tissue-engineered periosteum.

In vitro study of bioactivity of homemade tissue-engineered periosteum

This study was aimed to evaluate in vitro bioactivity of tissue-engineered periosteum (TEP) which was fabricated by coupling either rabbits' MSCs or osteodifferentiated MSCs (O-MSCs) with porcine small intestinal submucosa (SIS), and that produced two kinds of TEPs (M1=MSCs+SIS and M2=Osteoblast+SIS). The cell adherence was observed under scanning electronic microscopy (SEM). Cell proliferation was measured by 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-2 H-tetrazoliumbromide (MTT) test. In vitro osteogenetic potential of TEPs was evaluated kinetically at 0, 5, 10 and 15d by measuring alkaline phosphatase (ALP) activity, and semi-quantitative assay of osteocalcin and collagen type I expression with Western blot. SEM indicated that either MSCs or O-MSCs could adhere and survive on SIS, but MTT test revealed that SIS was more preferable for O-MSCs than MSCs in proliferative aspect. In contrast, ALP activity, osteocalcin and collagen type I expression of M2 were higher than M1 in general. Kinetically, osteocalcin and collagen type I expression demonstrated a continuous increase, while ALP activity of both groups was displayed an early peak around the fifth day, followed a decrease trend. In conclusion, TEP, especially M2, has an active osteogenesis and is promising for in vivo bone defect reparation via a supposed biomimetic procedure of intramembranous ossification.

The effect of mesenchymal stem cell sheets on structural allograft healing of critical sized femoral defects in mice

Structural bone allografts are widely used in the clinic to treat critical sized bone defects, despite lacking the osteoinductive characteristics of live autografts. To address this, we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets, which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates, as a tissue-engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets, suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo, allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of X-ray plain radiography, 3D microCT, histology, and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation, as well as graft-host osteointegration. Moreover, a large periosteal callus was observed spanning the allografts with MSC sheets, which partially mimics live autograft healing. Finally, biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together, MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair, demonstrating the feasibility of this tissue engineering solution for massive allograft healing.