WNT3A Regulates Osteogenic Differentiation of Periosteum Cells in HA/TCP Matrix

Diwei Wu^1#, Shaoyu Liang^1#, Xuanhe You^1#, Jiazhuang Xu^2* & Shishu Huang^1*

¹Department of Orthopedic Surgery, West China Hospital, Sichuan University, Chengdu, China
²College of Polymer Science and Engineering and State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, China

# Authors have equal contribution

*Correspondence to: Dr. Shishu Huang & Jiazhuang Xu, Department of Orthopedic Surgery, West China Hospital & College of Polymer Science and Engineering and State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, China.

Copyright © 2019 Dr. Shishu Huang & Jiazhuang Xu, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction
Bone engineering has been considered as one of the most important strategies in bone regeneration. The generation of new bone is aimed to replace or restore the dysfunctional or damaged bone. This meets the need of clinical and socio-economic interests [1]. Combination of cells, scaffolds, and appropriate growth factors is one of the methods in the regeneration of bone defects. These factors include BMPs, FGFs, VEGFs, IGFs, TGF-beta, PDGF, PTH/PTHrP, etc. Up to date, the generation of new bone has not yet been efficient and it requires further optimization [2]. In particular, Wnt3a, as one of the growth factors, has been highlighted in the involvement of bone development and generation. However, the role of Wnt3a in bone regeneration is still not clear.

Periosteal cells are a heterogeneous population that has a potential of osteogenic differentiation, which contains osteogenitor cells [3-5]. Periostem cells have been reported to regenerate bone defects, which are believed to mimick periostem for bone repair [3-5]. However, Seeding periosteum cells for bone issue engineering is a challenge that the osteogenetic capacity in vivo is still not efficient. HA/TCP matrix is regarded as one the promising materials in bone replenish [6,7]. It has been reported to be osteogenic induction [6,7]. Wnt family proteins are generally involved in the regulation of vertebrate skeletal development and bone homeostasis [8,9] Wnt3a, one of the nineteen members, was identified as a key factor in the development of long bones. Wnt3a was associated with Wnt4 and Wnt14 to activate the canonical Wnt signaling pathway in the developing synovial joints [10].

In this study, we hypothesize that Wnt3a enhances new bone generation in vivo and has a potential treatment in bone defect. A population of Wnt3a over-expressed (Wnt3a OE) periosteum cells was generated and implanted in a calvarial defect mouse model [6]. Compared to periosteum cells, osteogenesis in vivo was increased in Wnt3a OE periosteum cells that were confirmed by Masson and micro-CT. This has further been used in the treatment of the calvarial defect mouse model that suggested a new bone formation was enhanced by the Wnt3a modulation. Together with tissue engineering and cell therapy, this study may shed new lights on bone regeneration.

Methods and Material

Cell Culture
All animal experiments were approval by the Institutional Animal Care and Use Committee, Shenzhen People Hospital, Jinan University. The Green Fluorescent Protein (GFP) mice (C57BL/6-Tg(UBCGFP) 30Scha/J) and C57 mice (4-week-old, 15~20g weight, male) were purchased from Dashuo laboratory animal Co., Ltd. The periosteal cells were harvested by enzyme digestion (contained 3mg/ml Collagenase Type I and 4mg/ml dispase) and cultured in minima essential medium (MEM) supplemented with 20% fetal bovine serum (FBS). The primary cells were cultured for 14 days before transfection and seeded in the density of 10 cells/cm².

Flow Cytometry Analysis
The GFP periosteal cells were trypsinized and washed with PBS twice. These cells were mixed with1ml cold 4-Phenylbutyric acid (PBA) and centrifuged. Subsequently, cells were mixed with antibodys (1 ul CD105- 647Flour, 1ul Scal-1-PE, 1ul CD34-FITC, 1ul CD45-APC Cytm7) and incubated in dark for 1.5-2h at 4˚C according to the manufacturer’s protocol. Cells (106 per unit) were subjected to flow cytometry (FACS) on a FACS Calibur and analyzed using FlowJo software. Three independent times of FACS were carried out for analysis.

Recombinant Adenovirus Vectors
Briefly, PCR-amplified mouse Wnt3a was subcloned into the vector pHBLV-CMV-EF1-RFP (Fig. 1) using the primer pair Wnt3a-EcoRI-F (5’acacgaattcATGGACAGAGCGGCGCTCC3’) and Wnt3a- BamHI-R (5’acacggatccTTACTTACACGTGTGGACAT3’). The PCR products formed were confirmed by sequencing. The vector has been bought from HanBio company, Shanghai, China.

Lentivirus Packaging and Infection
HEK293T cells were cultured in DMEM with 10% FBS under a humid atmosphere containing 5% CO₂ at 37ºC and reached 70-80% confluence for transfection. The target recombinant adenoviral plasmid and the auxiliary packaging original vector plasmid (pSPAX2 and pMD2G) were amplified in 293T cells by liposomes. After 48hrs transfection, the virus-containing medium was collected through the centrifuge of 72000g/min for 120min at 4ºC. The supernatants contained wnt3a-GFP lentiviruses and GFP lentiviruses that infected periosteal cells at 37ºC in CO₂ incubator. The transfection efficiency of lentiviruses were detected by FACS.

The Effects of Wnt3a in Bone Information in Vivo
Wnt3a OE periosteal cells (107~108) were seeded on HA/TCP matrix (Wnt3a OE-cell-scaffold complex) and underwent subcutaneous transplantation on C57 mice (12-years-old, 25-30g weight, n=6). After two months transplantation, immunohistochemiscal staining, hematoxylin and eosin (H&E) staining and Masson trichrome staining were performed to evaluate the bone formation in vivo.

The Effects of Wnt3a in Bone Defect
Calvarial defect (5mm in diameter) mouse models (12-years-old, 25-30g weight) were established and randomly divided three groups: Wnt3a OE group (n=9), Control group (n=9), Blank group (n=9) and implanted with Wnt3a OE-cell-scaffold complex, cell-scaffold complex and scaffold respectively (10). After two months transplantation, the defected calvarial tissues were stained with H&E, Masson staining and scanned by micro-CT to evaluate the regeneration of bone defect. Fractional bone volume (bone volume per tissue volume, BV/TV), trabecular number (Tb.N), and separation (Tb.Sp) were calculated.

Statistical Analysis
All quantitative data were analyzed by SPSS Statistics (v.3a.0) and the significance of differences was determined using the ANOVA. Values are expressed as mean±SD with significance level set to p<0.05.

Results

The Generation of Wnt3a Over-Expressed Periosteal Cells
We harvested the periosteal cells from GFP mice and detected the mesenchymal stromal cell markers by FACS. 46.3% cells expressed CD105 positively and CD34 negatively, 50.6% cells expressed Sca-1 positively and CD45 negatively (Fig. 2). About 50% cells harvested from periosteum expressed both CD105 and Sca-1. For the lentiviruse transfection in GFP periosteal cells, about 22% population of periosteal cells were Wnt3a positive that were observed in yellow after overlap (Fig. 3). The FACS results further confirmed the over-expression of Wnt3a in Wnt3a positive cells (Fig. 4). The transfection rate was 22.1% that was consistent with the fluorescent microscope.

Wnt3a Promotes Atopic Bone Formation in Vivo
Wnt3a OE-cell-scaffold complex showed GFP positive after GFP immnunohistochemistry. Additionally, vessels, bone-like matrix and osteoblasts were observed in both complex after two-month implantation (Fig. 5). This suggested that periosteal cells and HA/TCP matrix promoted new bone formation in vivo. Compared to cell-scaffold complex, bone-like tissue was observed in Wnt3a OE-cell-scaffold complex and Wnt3a may enhance the osteogenesis of periosteal cells in vivo.

Wnt3a Associates Bone Regeneration in Calvarial Defect
C57 mice were underwent calvarial defect operation and behaved normally before implantation [6]. Calvarial defect mice were transplanted with HA/TCP matrix, cell-scaffold complex, and Wnt3a OE-cell-scaffold complex. All the three groups were implanted for two-month and harvested for microtomography (Micro- CT) that provides structural or architectural information about a bone sample. For the coronal plane of Micro-CT analysis, Wnt3a OE group showed superior conjunction of new lamellar bone with the calvarial defect, compared with the other two groups (Fig 6). The length of connection is the longest in the Wnt3a OE group. In the blank group, the gap between new lamellar bone and calvarial defect is larger than the other two groups. For the vertical plane, the bone density is highest in Wnt3a OE group compared with the control and blank group (Fig 6). For the cross section analysis, yellow parts indicate the bone tissue and mineralization was identified in the grafted scaffold by Micro-CT. New bone formation occurred around the edge of the calvarical defect in all the three groups and only Wnt3a OE group showed new bone formation in the center of the defect area (Fig.6). Interestingly, bony islands were observed in the defect areas of the control and Wnt3a OE groups. Additionally, the volume of the new bone, the average space of bone trabecular, and the average number of bone trabecular were measured by Micro-CT system. Wnt3a OE group significantly generated higher volume, more bone trabecular and lower trabecular space in new bone, compared with those in the control and blank groups. (Fig 6). By contrast, the control group indicated no significant differences in the average space of bone trabecular and the average number of bone trabecular compared with the blank group.

Histological Analysis of New Bone in Calvarial Defect
Histological analysis were underwent further and included hematoxylin-eosin (H&E) staining and Massoon staining. H&E staining showed lamellar new bone formation were observed in the calvarial defect in the three groups (Fig.7 A, B, C). Wnt3a OE group showed the largest areas of lamellar new bone formation compared to the other two groups. Masson trichrome staining identifies muscle, collagen fibers, fibrin and erythrocytes (Fig.7 D, E, F). This was performed in the three groups that the results were consistent with H&E staining. In the aspect of vessel formation, Wnt 3a OE group and the control group had higher rate of new vessel formation.

Discussion
Wnt3a is one of the Wnt family members and has been involved in the regulation of osteogenesis [11]. Wnt proteins belongs to a family that secret cystein-rich glycoprotein and are regulated by Wnt canonical pathway (Wnt-β-catenin pathway) and noncanonical Wnt pathway [12,13]. Wnt3a has been reported in playing an important role in the cortical bone thickness and the risk of nonvertebal fracture in human beings. Wnt3a induced osteogenesis and inhibited osteoclastogenesis in mouse and human being. This modulated osteoclasts and promoted the osteoprotegerin (OPG) expression in osteoblasts [11]. Additionally, Wnt3a has been reported to be associated with bone mass and bone plasticity in a mouse model of osteogenesis imperfect [14]. In our study, Wnt3a has been involved in the osteogenesis of periosteum cells and in vivo bone defect regeneration model.

Wnt3a expression was upregulated in the perichondrium and periosteum in the development of skeleton [15]. Wnt3a was identified to be involved in the regulation of mouse craniofacial bone formation. Wnt3a expressed in the osteoid of calvaria, maxilla and mandible during intramembranous ossification. This process was between the expression of alkaline phophatase (ALP), osteocalcin (OCN) and the occurrence of bone mineralization [8]. Furthermore, the association between Wnt3a and bone mineral density (BMD), cortical bone thickness, bone strength and osteoporotic fracture risk has been proved by large population-based genome-wide association studies (GWASs) and in Wnt3a (Wnt3a-/-) knockout mouse model as well [16- 18]. The deletion of Wnt3a caused cortical bone thickness and porosity and didn’t affect trabecular bone mass. The up-regulation of Wnt3a promoted Opg expression in osteoblasts that contributed to inhibit osteoclastogenesis by suppressing RANKL-induced activation of nuclear factor-κB (NF-κB). It has been suggested that Wnt3a might act as a critical regulator for the communication between osteoblast and osteoclast in bone homeostasis [18]. In our study, we investigated the effects of Wnt3a involved in bone formation in vivo by using mouse model and discovered that there were more vessels, bone-like matrix and osteoblasts observed in Wnt3a OE group, compared to control group. This suggested that Wnt3a might promote the osteogenic differentiation and contribute to the bone formation. We provide the evidence that Wnt3a might be also a key regulator in the promotion of osteogenesis. Additionally, other growth factors have also been reported in the involvement of bone formation. These factors include Vitamin K, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derivedgrowth factor- 1alpha (SDF-1α), Insulin like growth factor-1 (Igf-1).

Nowadays clinical and socio-economic requirements are highlighted in the use of new bone to replace or maintain the function of damaged, traumatized or lost bone [2]. Tissue engineering and regenerative medicine shed a new light on the bone repair and regeneration. The discipline of tissue engineering in bone regeneration is in the combination of cells with scaffolds, or with growth factors, by which initiates and promotes osteogenesis. In the study, we used periostem cells, osteo-inducible scaffolds, and biological factor Wnt3a to create a robust and biocompatible bone regeneration strategies to repair the bone defect. This is targeted to address the requirement for bone regeneration and skeletal repair.

In this study, we choose the mouse calvarial defect model for the study of bone regeneration, which is a convenient model and has less risk in the occurrence of complication [19]. A 5-mm calvarial bone defect has been considered as a critical-size defect and the application of the HA/TCP scaffold not only provided with physical support but also extracellular microenvironment. As a supportive platform, the material scaffold has advantage and disadvantage, which is analysis along with various architectural parameters, including interconnectivity, porosity, pore size and pore-wall microstructures [1].

After two months of implantation, all the calvarial defect mice were investigated by both Micro CT and histological analysis, which were used widely and reliable in the assessment of bone regeneration [20]. Micro CT showed that new bone formation in the defect border was observed between the implanted sites and the host bone in all the three groups. In the blank group, the proliferation of host bone cells and the physical support of the scaffold contributed to the new bone formation. In the control group, the periostem cells divided into osteoblast and more volume of trabecular bone were regenerated compared to the blank group. Interestingly, new bone was regenerated into deeper parts of the scaffold and bony islands scattered in the defect areas in the Wnt 3a OE group. This indicated Wnt3a derive osteoblast in vivo [21]. In our study, Masson trichrome staining demonstrated that a larger number of vascular blood formation in the control and Wnt3a OE groups, which resulted from the combination of the scaffold with periostem cells (Fig.5 C, D). The establishment of a vessel blood network is the key of tissue engineering. The vasculogenesis and angiogenesis are provided with nutrition supply, gaseous exchange and waste removal during the new bone formation [2,22-24].

Conclusions
The results of this study indicated that the use of periostem cells enhanced bone defect regeneration and the Wnt3a OE promoted bone formation and mineralization. Moreover, the establishment of blood vessel network was also observed. The combination with the HA/TCP scaffold and Wnt3a provided a osteogenesislike microenvironment, which promoted the bone repair and regeneration in the calvarial defect in vivo.

Acknowledgements
The authors declare that there is no conflict of interest regarding the publication of this paper.

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