Mineralized Collagen-Based Composite Bone Materials for Cranial

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Mineralized Collagen-Based Composite Bone Materials for Cranial Bone Regeneration in Developing Sheep Shuo Wang, Yongdong Yang, Zhijun Zhao, Xiu-Mei Wang, Antonios G. Mikos, Zhiye Qiu, Tianxi Song, Xiaodan Sun, Lingyun Zhao, Chunyang Zhang, and Fu-Zhai Cui ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 26, 2017

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ACS Biomaterials Science & Engineering

Mineralized Collagen-Based Composite Bone Materials for Cranial Bone Regeneration in Developing Sheep Shuo Wang†#, Yongdong Yang†#, Zhijun Zhao‡, Xiumei Wang†*, Antonios G. Mikos§, ⊥ ⊥ Zhiye Qiu , Tianxi Song , Xiaodan Sun†, Lingyun Zhao†, Chunyang Zhang‡*, Fuzhai Cui† † State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China. ‡ The First Affiliated Hospital of Baotou Medical School, Department of Neurosurgery, Baotou 014010, China § Department of Bioengineering, Rice University, Houston, TX 77030, USA ⊥ Beijing Allgens Medical Science and Technology Co., Ltd., Beijing 100176, China * Corresponding author: Xiumei Wang, E-mail: [email protected]; Fax: +86-10-62771160; Tel: +86-10-62782966 Chunyang zhang, E-mail: [email protected]; Tel: +86-13030480336

# These two authors contribute to this work equally. Full Mailing Address of Authors 1. Shuo Wang, Yongdong Yang, Xiumei Wang, Xiaodan Sun, Lingyun Zhao, Fuzhai Cui YiFu Science and Technology Building, No.1 Qinghuayuan, Tsinghua University, Haidian, Beijing 100084, China. 2. Zhijun Zhao, Chunyang Zhang The First Affiliated Hospital, No.41 Linyin Road, Kun District, Baotou 014010, China. 3. Antonios G. Mikos Rice University, MS-142, P.O. Box 1892, 6500 Main Street, Houston, TX 77030, USA 4. Zhiye Qiu, Tianxi Song

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ABSTRACT

Cranial bone defects remain a great challenging problem in clinic, the influences of which are serious due to the intricate complications and related social problems, especially for young children with rapidly growing skulls. Currently, an increasing number of bone materials are being developed for cranial bone defects repair. In this study, two different biodegradable composite bone materials based on mineralized collagen (MC), with compact/porous structure, were constructed to promote bone regeneration for large cranial bone defect repair of one-month-old baby sheep. The porous MC (pMC) scaffold had interconnected porous structure with a porosity of about 73% and a 20-150 µm pore size range, and the compact MC (cMC) showed no distinct pore structure. Mechanical test indicated that the compressive strength and elastic modulus of cMC and pMC were comparable with those of natural compact and cancellous bone, respectively. Both of these two MC scaffolds possessed good biocompatibility and supported osteoblasts adhesion and proliferation in vitro. A One-month-old sheep cranial bone defect model was firstly established to investigate the cranial bone regeneration behaviors in vivo, which was evaluated by CT imaging, X-rays scans as well as histological assessments. It was found that the pMC promoted bone ingrowth from the diploic layer of surrounding cranium and dura mater-derived osteogenesis at three months after surgery, along with gradual biodegradation. In contrast, the cMC had very little biodegradation but could promote bone formation beneath the scaffold through dura mater-derived osteogenesis pathway. Furthermore, Ti-mesh restricted the growth of surrounding cranial bone in the rapidly growing



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sheep, thereby causing obvious deformation of the skull at six months after surgery. While no visible geometric deformation of skull occurred in the cMC and pMC groups. Our findings suggested that the MC-based composite bone materials have great promise for the repair of large cranial bone defect in a developing skull.

KEYWORDS：： mineralized collagen; cranial bone; bone regeneration; developing

sheep; bone tissue engineering


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1. INTRODUCTION

Cranial bone defects, which can occur as a result of congenital defects like dysraphism and skeletal anomaly or acquired injuries including trauma, tumor, encephalic and maxillofacial surgeries, and infection, usually result in severe complications, such as cranial bone defect syndrome, infection, brain swelling, hydrocephalus, epilepsy, or hemiplegia, and sometimes even lead to psychological and social problems.1-3 Therefore, adequate skull reconstruction in both functional and cosmetic ways is of vital importance for patients to provide stable biomechanical support, enough cerebral protection, and optimal cosmetic results. Especially for young children with rapidly growing skulls, timely and effective repair is thought to be critical for their healthy growth.4-5

In clinic, the commonly used repair method, called cranioplasty, is a general surgical intervention to repair skull defects or deformity with biomaterials, which is dominantly performed on adults with large-size bone defect.6-7 Many different types of materials have been used for cranioplasty, including autograft, allograft, xenograft as well as artificial substitutes. Autograft is harvested from patients’ own bone tissues (autologous bone), such as tibia, rib, scapula, fascia, sternum, or ilium. Autologous bone has excellent osteoconductivity and therefore has been widely recognized as the “gold standard” for healing large bone defects. However, lots of serious problems associated with autografts couldn’t be disregarded. In addition to donor-site morbidity and limited graft quantity, it’s hard to shape autologous bone to match the cranial



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contour and cover the defected skull sufficiently. As for allograft or xenograft, it couldn’t be shaped either and has the potential risk of transmitting disease and the immunological response at the same time. Therefore, a variety of artificial materials such as titanium, bio-glass, polymethyl methacrylate (PMMA), polyetheretherketone (PEEK), and hydroxylapatite have been developed for cranioplasty. However, none of these artificial substitutes mentioned above are satisfying. For example, titanium is the most common used cranioplasty material, but it produces significant image artifacts in CT and MRI imaging, and may damage brain tissue because of high heat conduction. Furthermore, it should be noted that all these currently used cranioplasty materials are non-biodegradable, which will result in restricted growth and deformation of the skull especially for pediatric patients whose skulls are growing.5,8 Therefore, skull reconstruction for young children remains a great challenge in clinic. There is still a widespread controversy on the timing in surgery.5,9,10 Because the cranioplasty techniques applied in clinic nowadays only fill up the defect with implants to repair in shape instead of the regeneration of cranial bone, developing new cranioplasty materials to induce cranial bone regeneration could probably provide a new way to subside the controversy. Currently, an increasing number of biodegradable materials have been explored to regenerate cranial bone defect, while most of them are far from the clinic because of the unignorable disadvantages, such as the lack of appropriate mechanical support or limited osteogenic activity.7, 11-14 An ideal cranioplasty material must fit the cranial defect and achieve complete closure with good biomechanical properties, radiolucency, resistance to infections, low heat


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conductivity, as well as outstanding osteoconductivity, biodegradability, and conformability.15-19

Cranial bone regeneration is challenging, because the regeneration rate of flat bones like calvarial bone, ilium, and rib were much slower than that of long bones owing to the different ossification processes, intramembranous ossification (flat bone) and endochondral ossification (long bone).20 Previous reports have shown that three pathways could be involved in cranial bone regeneration. The first pathway is the osteogenesis through the new bone invasion from the diploe, which is a layer of cancellous bone containing limited blood vessel system between two thin layers of compact bones of cranial bone. The second pathway is from the periosteum located on the outer surface of cranial bone, which contains progenitor cells/osteoblasts to deposit new bones. Besides, the outmost periosteal layer of dura mater serves as the skull’s inner periosteum that has the similar physiological function of inducing new bone formation.21-24 However, periosteum is usually broken or even absent during cranial injures, as a result of which, the cranial bone repair material had better possess the ability to promote the bone regeneration through the diploe and/or the dura mater pathway. Besides, the cranial bone repair materials should also have appropriate mechanical properties like bending strength and toughness to provide enough protection and keep a stable intracranial environment.

In this study, we developed two kinds of biodegradable bone materials based on mineralized collagen (MC), compact MC and porous MC composite materials. MC is



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developed previously in our laboratory via in vitro biomimetic mineralization process, which mimics the composition of the nature bone, showing similar hierarchically self-assembled microstructures.25-26 MC has already been widely applied in long bones repair and spinal fusion, displaying excellent biocompatibility and osteogenic capability.27-30 Here, one-month-old baby sheep cranial bone defect model was firstly established to evaluate the performance of bone materials on promoting the regeneration of cranial bones at a rapidly growing period.

2. MATERIALS AND METHODS

2.1 Preparation of the MC-based composite scaffolds

MC powder was prepared as previously described. In brief, type I collagen was dissolved in dilute HCl to form an acidic collagen solution. Then CaCl2 solution and NaH2PO4 solution (Ca/P=1.67) were dropwise added to the collagen solution in turn with stirring. The pH value of the mixed solution was then adjusted to 7.4 with NaOH solution and gently stirred for 48 h. The MC deposition was gradually formed and then harvested by centrifugation. After washing in deionized water for three times, MC was lyophilized and milled into powder for use.

The porous MC composite scaffold (pMC) was prepared by mixing MC powder with biodegradable polymer, polycaprolactone (PCL) according to the following procedure. PCL (Jinan Daigang Biomaterial Co., biomedical grade) was dissolved with 1,4-dioxane. MC powder was then added to the PCL solution at a weight ratio of 1:1 with stirring to form a homogenous slurry. The slurry was then poured into a


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cylindrical mold and stored in -20°C overnight, followed by a freeze-drying to remove the organic solvent. After

60

Co irradiation sterilization, the scaffolds were

preserved in sterilized condition.

The compact MC composite scaffold (cMC) was prepared by mixing MC powder with melted PCL according to the following procedure. PCL was heated to about 80°C in a viscous state under which a slight force could change its shape. The same weight ratio of MC powder was added into the viscous PCL and mixed homogeneously by mechanical force. Then the mixture was molded to the final shape of scaffold by cooling to room temperature.

2.2 Measurements on the physical properties of scaffolds

Scanning electron microscopy (SEM) was used to show the microstructures of pMC scaffold as well as the distribution of pore size. The porosity of pMC scaffold was determined via a method of liquid displacement with water (ρ=1.0 g/cm3), in which materials with regular shapes were used (the volume can be known as v1) and the volume of absorbed water (v2) equaled to that of pores, so that the porosity equals to v2/v1 ⨉ 100%.

The compressive strength and elastic modulus of the pMC scaffolds as well as the cMC scaffolds were measured with a 250 N load cell using universal mechanical testing machine (SHIMADZU AG-IC, Japan). The samples were prepared as standard cylinders with 10 mm in diameter and 20 mm in length. The load was applied until the pMC scaffold was compressed to approximately 30% of its original length. The



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compressive modulus was calculated as the slope of the initial linear portion of the stress-strain curve. The compressive strength was determined as the intersection with the line from the 20% strain point with a same slope of elastic modulus. Three individual samples from each type of bone material were repeatedly measured for statistical analysis.

2.3 Cells culture and in vitro biocompatibility evaluation

The mouse osteoblast cell line (MC3T3-E1, passage 6-8, Cyagen Biosciences Inc.) were cultured in low glucose Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin Solution (PS) under a humidified atmosphere consisting of 95% air and 5% CO2 at 37°C

In vitro biocompatibility of the MC-based scaffolds were evaluated by cell culture of MC3T3-E1. Briefly, the pMC and cMC scaffolds were molded into round disks with 30 mm in diameter and 3 mm in thickness. The MC3T3-E1 were seeded onto the prepared pMC and cMC round disks in a 6-well cell culture dish at a concentration of 1×105 cells/cm2. After 12 h of cell culture in the regular growth medium, cell adhesion was examined using SEM. The specimens were fixed with 2.5% glutaraldehyde in Phosphate Buffered Saline (PBS, 0.1M, pH=7.4), followed by gradient dehydration from 30% ethanol to 100% ethanol. The samples were finally dried through critical point drying (Samdri-PVT-3D, America). The surfaces of the samples were further sputter-coated with a layer of platinum for SEM observation


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using Field Emission Scanning Electron Microscope (Zeiss, Germany).

In order to investigate the cell proliferation on the MC scaffolds, the MC3T3-E1 were seeded at a concentration of 2.5×104 cells/cm2 and cultured for up to 7 days. The pMC scaffolds were molded into round disks with a diameter of 10 mm. For evaluating the effect of the MC composition on cell proliferation and avoiding the influence of porous structure, the pMC round disk was pressed into a compact state. After 1 day, 3 days, or 5 days of cell culture, the samples were taken out and fixed with 4% paraformaldehyde in PBS and stained with rhodamine for skeleton protein as well as DAPI for nucleus. The morphologies were visualized through Laser Scanning Confocal Microscopy (LSCM, Zeiss LSM710, Germany) to show the proliferation qualitatively as well as general status of cells. A furthermore quantitative evaluation of cell proliferation was realized by Cell Counting Kit-8 (CCK-8, Dojindo, Japan) for two kinds of cells culture situations, on the pressed pMC round disks and in leaching solution. The leaching solution was made by retrieving the complete cell culture medium with the MC round disk immersed in it for 3 days.

2.4 In vivo evaluation using a sheep cranial bone defect model

A critical-sized cranial bone defect (3 cm in diameter) model was used to evaluate the bone regeneration under the bone materials repair. Totally eight healthy 1-month-old sheep were used in this study. The sheep were randomly divided into four groups of 2 animals each: defect without implantation (blank group), pMC scaffold implantation (pMC group), cMC scaffold implantation (cMC group), and titanium mesh



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implantation (Ti-mesh group). The surgeries were carried out at the First Affiliated Hospital of Baotou Medical College, China.

Under a general anesthesia by the injection of 3% sodium pentobarbital (30 mg/kg weight), the sheep were shaved and the incisions were made upon the calvarias with the periosteum partially removed to expose the cranial bones. A 30-mm-diameter round defect in the center of calvaria was then drilled by bone drill as well as rongeur forceps, keeping the dura mater intact (Figure 1a). After the implantation of pMC, cMC or Ti-mesh (Figure 1b), the wound was sutured (Figure 1c). The pMC, cMC and Ti-mesh were fixed on the defects by direct suture, plastic connection pieces, and screws, respectively. After the surgeries, 1600,000 IU penicillin was injected through intramuscular to the sheep once a day for 5 days.

Computed Tomography (CT) scans were taken immediately post-surgery and 3 months after the operation in order to get three-dimensional (3D) reconstructed images through which the status of cranium regeneration could be observed. Besides, the CT reconstructed images for cMC group and Ti-mesh group were obtained 6 months after operation to compare the geometric deformation of peripheral cranium. X-rays scans were also taken at 2 months after the surgery as an assistant evaluation of osteogenesis according to the difference of density on images.

Samples of calvarial bone containing both the implants and surrounding original cranial bone were harvested at 3 months after operation for further histological evaluation. The calvarial bone samples collected from the pMC group were fixed with


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4% formaldehyde for 12 h and decalcified with 10% ethylenediaminetetraacetic acid (EDTA) for 6-8 weeks. After that, the tissue blocks were embedded in paraffin and cut into 4-µm-thickness tissue slices using Microtomes (Leica RM2234, Germany). Both the Masson’s trichrome staining and Hematoxylin-Eosin (H&E) staining were conducted for histological evaluation using a light Microscope (OLYMPUS IX81, Japan).

2.5 Statistical analysis

All numerical data are reported as the mean ± standard deviation. The statistical analysis was performed with one-way analysis of variance and Student’s t-test. The data were considered statistically significant when p < 0.01. All of the data were analyzed using SPSS 13.0 software for Windows, Student Version.

3. RESULTS AND DISCUSSION

3.1 Structural and mechanical properties of MC scaffolds

The two types of MC scaffolds, pMC and cMC were shaped into round disks with diameters and thicknesses of 3 cm and 3 mm, respectively. The gross morphologies of pMC and cMC applied in the in vitro implantation were shown in Figure 2a. The typical SEM morphology of pMC was shown in Figure 2b, exhibiting interconnected pore structure with size distribution between 20 µm and 150 µm. And the porosity of the pMC was calculated to about 73.6 ± 2.0%. Additionally, it could be clearly seen that the MC particles scattered homogenously in the PCL, as shown in the high



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magnification SEM images of pMC (inserted image in the top right corner of Figure 2b). In contrast, cMC showed no any pore structure instead of compact and homogeneous PCL phase containing uniformly scattered MC particles (Figure 2b).

The mechanical properties of the MC scaffolds were tested and determined by the compressive stress-strain curves. As shown in Table 1, the compressive strength and elastic modulus of cMC (σ = 32.63±0.75 MPa, E = 0.59±0.01 GPa, p

Mineralized Collagen-Based Composite Bone Materials for Cranial

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