Bone regeneration in minipigs by intrafibrillarly-mineralized collagen loaded with autologous periodontal ligament stem cells

Biomimetic intrafibrillarly-mineralized collagen (IMC) is a promising scaffold for bone regeneration because of its structural and functional similarity to natural bone. The objective of this study was to evaluate the bone regeneration potential of IMC loaded with autologous periodontal ligament stem cells (PDLSCs) in large bone defects in minipigs. A macroporous IMC with a bone-like subfibrillar nanostructure was fabricated using a biomimetic bottom-up approach. Non-healing full thickness defects were established on the cranial bone in minipigs, and IMC and hydroxyapatite (HA) scaffolds seeded with autologous PDLSCs were implanted into these defects. Computed tomographic imaging, histology staining, and atomic force microscopy were applied to evaluate to the quantity, micro/nano structures, and mechanical performance of the neo-bone after 12 weeks of implantation. Compared with HA, IMC showed superior regeneration properties characterized by the profuse deposition of new bony structures with a normal architecture and vascularization. Immunohistochemistry showed that the runt-related transcription factor 2 and transcription factor Osterix were highly expressed in the neo-bone formed by IMC. Furthermore, the nanostructure and nanomechanics of the neo-bone formed by IMC were similar to that of natural bone. This study provides strong evidence for the future clinical applications of the IMC-based bone grafts.

Isolation and identification of minipig PDLSCs. The minipig PDLSCs were isolated and cultured under the same conditions employed in our previous study for human PDLSCs 26 . Periodontal ligament tissue was scraped from the middle third of the root surface of the incisors in minipigs and was digested with collagenase type I (3 mg/mL; Worthington Biochemical, USA) and dispase II (neutral protease, 4 mg/mL; Roche Diagnostics, USA) for 2 h at 37 °C to obtain a single-cell suspension. After isolation, the cells were cultured in alpha minimum essential medium (Gibco, Thermo Fisher Scientific, Waltham, MA) with 20% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT), 100 U/mL penicillin/streptomycin, 2 mM glutamine, 55 mM 2-ME (Gibco), and 0.1 mM L-ascorbic acid phosphate (Wako Chemicals, Richmond, VA) at 37 °C in 5% CO 2 . The 3rd passage of PDLSCs was used in the following experiments. The multipotency of the PDLSCs was confirmed by examining osteogenicity using Alizarin red S staining and adipogenicity using Oil red O staining (Fig. S1).
In vitro cell seeding on different scaffolds. To examine the influence of scaffolds on cell growth and extracelluar matrix (ECM) secretion, minipig PDLSCs at passage 3 were seeded at 1 × 10 6 cells/scaffold and were cultured with regular medium without any osteogenic supplements at 37 °C in 5% CO 2 . Before cell-seeding, the scaffolds were sterilized with 75% ethanol for 24 hours and were immersed in PBS with 100 U/mL penicillin/streptomycin overnight. On days 3 and 7, the scaffolds were fixed with 2.5% glutaraldehyde in PBS and lyophilized for SEM examination.
Animal surgery. Two non-healing full thickness defects of approximately 2 cm width × 3 cm length × 0.5 cm depth were established on the cranial bone of each minipig and eighteen defects were randomly divided into experimental and control groups. The IMC (N = 6) and HA (N = 6) scaffolds (Dongbo Biotechnology, China) seeded with 1 × 10 6 minipig PDLSCs were randomly placed into the defect area, and no implant was used in the negative control (N = 6). After 12 weeks of implantation, the minipigs were sacrificed via anesthesia overdose. The craniums were obtained from each group and fixed with 10% formalin in PBS after the removal of soft tissue.
Computed tomographic (CT) measurements. To evaluate the formation of new bones by different scaffolds, the fixed craniums were scanned using CT imaging (Siemens Medical Solutions, Knoxville, TN, USA) at 80 kV and 500 mA. The Inveon Research Workplace software (Siemens, USA) was applied to calculate the ratio of bone volume/tissue volume (BV/TV) and bone mineral density (BMD) in the defect area. To calculate BV/TV, the gray value was set between 400 and 1200 to eliminate the influence of the residual scaffold on bone volume. Histological analyses. After CT scanning, half of the craniums were decalcified in 15% EDTA for 8 weeks, dehydrated in a graded series of ethanol (70-100%) and embedded in paraffin. Serial tissue sections with 5 μm thickness were prepared from the mid-sagittal plane of the defect area, treated with hematoxylin-eosin (HE) and Masson's trichrome staining, and observed under a light microscope (Carl Zeiss Inc., Germany). The residual scaffold fraction was calculated as the residual scaffold area divided by the defect area using histomorphometric techniques. After Masson's trichrome staining, the blue color indicates the regenerated bone, collagen fibers, or osteoid, while the red color indicates the mature bone.
Atomic force microscope (AFM) measurements. The other half of the craniums were undecalcified and sectioned from the center of the defect areas. AFM (Dimension Icon, Bruker, USA) was applied to test the nanostructure and nanomechanics of the newly-formed bones under ambient conditions. Six regions of interest (ROIs) were selected, and the median value was used to represent the property value for each ROI. Thus, 18 values (3 samples × 6 ROIs) were generated for each group.
Statistical analysis. All data were expressed as mean ± standard deviation and assessed via one-way ANOVA and Holm-Sidak pairwise comparison after performing normality and equal variance tests. Statistical significance was considered at P < 0.05.
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).

Results
Characterization of scaffolds. The IMC was fabricated using a modified biomimetic bottom-up approach, in which collagen fibrillogenesis and nanoapatite platelet formation occurred simultaneously. After free drying, 3-D porous IMC scaffolds were obtained and showed a spongy morphology with interconnected macropores of 148.2 ± 46.5 μm (Fig. 1). Under high magnification, the collagen fibrils exhibited bone-like subfibrillar nanostructure with cross-banding patterns (Inset). No apatite crystals were formed around the fibrils, suggesting that the mineral phase is primarily embedded within the fibrils. The presence of nanoapatites within the collagen fibrils was revealed by EDS, in which the Ca/P ratio was 1.52 ± 0.06, indicating of calcium-deficient nanoapatites. The HA scaffold composed of micro-sized apatites showed ununiform macropores of 345.7 ± 47.4 μm and the Ca/P ratio was 1.61 ± 0.03.

Matrix secretion by PDLSCs on different scaffolds.
The primary minipig PDLSCs showed typical fibroblastic morphology with a spindle shape and formed evident clones after three days of culture. The positive staining of Alizarin red and Oil red confirmed the osteogenic and adipogenic differentiation ability of PDLSCs (Fig. S1). To observe cell-scaffold interactions, the identified PDLSCs were seeded on different scaffolds. After 3 days of culture, PDLSCs contacted well with the IMC and HA scaffolds via their filopodia. The cells were spread all over the surface of the IMC scaffold and secreted some ECM, whereas few cells without ECM secretion were observed on the surface of the HA scaffold ( Fig. 2A). After 7 days of culture, the PDLSCs seeded on the IMC developed cytoplasmic extensions with abundant cell-cell junctions, and produced numerous matrix vesicles that were attached to the cell membranes. By contrast, the PDSLCs seeded on HA scaffold showed a less extensive ECM secretion with large calcium nodules deposited on the cell surface (Fig. 2B).

CT analysis of new bone volume in minipigs.
To investigate the bone regeneration potential of the IMC scaffold in large animals, non-healing full thickness defects without periosteum were created in cranial bone of minipigs (Fig. 3A,B). After implantation for 12 weeks, these bone defects were almost filled with fibrous bone structures even at the defect center and the depth of such defects significantly decreased in the IMC group (Fig. 3D,E). By contrast, the defect area in the HA group remained highly radiopaque, and an obvious boundary was observed between the scaffold and defect margin (Fig. 3D,F). The mineral density for the HA group was higher than that for natural bones (Fig. S2), thereby indicating that a large number of HA scaffolds did not degrade in the defect area. For the negative control, only a small amount of neo-bone was formed long the defect margin (Fig. 3C,G). According to the quantitative analysis, the IMC group achieved a significantly higher extent of new bone formation (BV/TV = 45.2 ± 17.7%) than the HA (BV/TV = 29.3 ± 7.7%) and control (BV/ TV = 19.6 ± 3.4%) groups (Fig. 3H). Furthermore, the mineral density of the new bones in the IMC group was similar to that of natural bones. (Fig. S2).

Histological evaluation of regenerated bone in minipigs. Histological analyses including HE and
Masson's trichrome stainings were performed to observe the microstructure of tissues in the defect area (Fig. 4). In the IMC group, plenty of new bones with few unresorbed scaffold remnants were observed along the defect margin, and the appearance of osteon with osteocytes and blood vessels in the defect center indicating the active formation of neo-bone (Figs 4 and S3). By contrast, limited new bone structure with a large number of undegraded HA remnants was identified in the defect area in the HA group. The residual HA scaffold volume (40.88 ± 6.29%) was six times greater than the residual IMC scaffold volume (6.34 ± 2.14%) (Fig. S4). The remnants of HA scaffolds prevented the establishment of a completely normal architecture of natural bones and blood vessels. In the control group, only a small amount of new bone was seen along the defect area. Consistent with the CT data, the semi-quantitative analysis results (Fig. S3) indicate that the IMC group has a significantly greater amount of new bones than the other groups (P < 0.001).
The Masson's trichrome staining of the defect center showed that abundant new collagen fibers and a completely normal architecture of natural bone with osteocytes and blood vessels could be observed in the IMC group (Fig. 4). Some mature bones with red dye were also observed in the IMC group. The limited number of new bones with red dye in the HA group showed a relatively high maturity, yet lacked the normal architecture of natural bones. A small amount of new bones with blue dye was observed in the control group.
Runx2 and Osx are two early transcription factors required for osteoblastogenesis and bone formation 32 . From the immunohistologic staining, Runx2 and Osx were highly expressed in the IMC group, whereas weak or negative staining was observed in the HA group (Fig. 5). These findings indicate that more new bones (Fig. S3) and osteocytes (Fig. S5) are present in the IMC group than in the HA group. The expression level of TGF-β1, which influences growth, differentiation and ECM secretion during bone development 33 was also higher in the IMC group than in the HA group. The increased expression of TGF-β1 may contribute to the formation of ECM in the IMC group in vitro. Nanostructure and nanomechanics of newly-formed bone. The nanostructure and nanomechanics of newly-formed bone in the defect center in the IMC and HA groups were evaluated by AFM using the PeakForce QNM mode (Fig. 6). Mineralized collagen fibrils with distinct cross-banding patterns were identified in the IMC group and were similar to the nanostructure of natural bones. From the 3-D property maps, the modulus distribution of the newly-formed bones in the IMC group was similar to that of natural bones. The quantitative analysis also showed that the new bones formed in the IMC group (5.1 ± 0.7 GPa) possessed improved nanomechanical properties than those formed in the HA group (3.6 ± 0.8 GPa). This might be because that HA induced less neo-bone formation with many voids in the defect center.

Discussion
The introduction of novel biomaterials for human bone repair requires preclinical safety testing using laboratory animals. With bone anatomy, microstructure, and remodelling process that are similar to those of humans, minipigs have been used as large animal models for the pivotal preclinical testing of human skeletal implants 29-31 . In this study, we first reported the use of biomimetic IMC in combination with autologous PDLSCs for the regeneration of large bone defects in minipigs. Compared with rat critical-sized mandibular defects of approximately 5 mm in diameter in our previous studies 27,28 , the defect area of approximately 2 cm width × 3 cm length × 0.5 cm depth) in this study is an order of magnitude greater. The IMC still possesses an excellent bone regeneration and vascularization potential. Furthermore, the nanostructure and nanomechanics of the new bone generated in the IMC group were similar to those of natural bones, while the HA promoted disorganized formation of new bones  with poor mechanical properties. These measurements by AFM are critical for assessing new bone biofunctions because the bone collagen fibril architecture dramatically affect bone mechanical strength (e.g. osteoporosis) 34 .
Optimum bone grafts have three prerequisites, namely, compatibility with the surrounding tissue, controllable degradation rate, and proper porosity to protect the healing space and allow cell migration and blood vessel ingrowth [35][36][37] . The cell seeding experiment in this study showed that the porous IMC had an excellent biocompatibility and was suitable for minipig PDLSC attachment and proliferation. To achieve maximum bone generation, we should balance both degradation rate and healing time. After 12 weeks of implantation, the IMC almost degraded and facilitated an abundant new bone ingrowth, while numerous undegraded HA inhibited bone healing and normal bone architecture formation. Although HA, as a main inorganic component of natural bone, seems to be an ideal bone graft, its uncontrollable degradation rate remains a challenge 11,38 . The HA remnants also increased the volume and mineral density of the regenerated area evaluated by CT, thereby underscoring the importance of histological analysis for assessing new bone formation. Observing the bone regeneration potential of IMC and the degradation rate of HA requires a longer time in our future study. Other criteria for porous scaffolds include porosity and pore interconnectivity to allow cell infiltration and vascularization 39 . The optimal pore size for new bone ingrowth ranges between 100 microns and 400 microns 40 . The IMC has a proper pore size of 148.2 ± 46.5 μm and a more interconnective space than HA, both of which can lead to vascular tissue ingrowth in the IMC group.
Bone regeneration is essentially a process of ECM formation and mineralization 41 . Coaxing appropriate cell-material interactions toward new bone regeneration is a basic premise of biomaterials in tissue engineering. In this study, we demonstrated that the physical and chemical properties of scaffolds could affect the ECM secretion of seeded cells. Limited ECM with calcium nodules was formed on the surface of those cells that were seeded on the inorganic HA scaffold. By contrast, those cells seeded on the IMC composite secreted large amount of membrane-bound matrix vesicles with abundant cell-cell junctions, that resembled the matrix production process of bone formation in vivo 42 . The active ECM secretion in vitro may further contribute to bone regeneration in vivo. The increased expression of Runx2 and Osx during the bone regeneration process reflects the osteoinductive potential of IMC, which activates more bone-forming cells. TGF-β1 is an important transcription factor for regulating ECM formation and mineralization 43 . The increased expression of TGF-β1 in the IMC group suggests the involvement of the TGF-β1 signaling pathway in the formation of mineralized ECM as induced by IMC.

Conclusions
This study is the first to use biomimetic IMC in combination with autologous PDLSCs for the regeneration of large bone defects in minipigs. Compared with HA, IMC achieved a significantly higher extent of forming new bones, with the normal architecture of natural bones and blood vessels. The new bones generated in the IMC group also showed the similar nanostructure and nanomechanics to natural bones. Therefore, IMC presents a great potential for treating large bone defects in the future.