Graphene oxide-modified silk fibroin/nanohydroxyapatite scaffold loaded with urine-derived stem cells for immunomodulation and bone regeneration

Background The invasive and complicated procedures involving the use of traditional stem cells limit their application in bone tissue engineering. Cell-free, tissue-engineered bones often have complex scaffold structures and are usually engineered using several growth factors (GFs), thus leading to costly and difficult preparations. Urine-derived stem cells (USCs), a type of autologous stem cell isolated noninvasively and with minimum cost, are expected to solve the typical problems of using traditional stem cells to engineer bones. In this study, a graphene oxide (GO)-modified silk fibroin (SF)/nanohydroxyapatite (nHA) scaffold loaded with USCs was developed for immunomodulation and bone regeneration. Methods The SF/nHA scaffolds were prepared via lyophilization and cross-linked with GO using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS). Scaffolds containing various concentrations of GO were characterized using scanning electron microscopy (SEM), the elastic modulus test, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectrometer (XPS). Examinations of cell adhesion, proliferation, viability, morphology, alkaline phosphatase activity, and osteogenesis-related gene expression were performed to compare the osteogenesis-related biological behaviors of USCs cultured on the scaffolds. The effect of USC-laden scaffolds on the differentiation of macrophages was tested using ELISA, qRT-PCR, and immunofluorescence staining. Subcutaneous implantations in rats were performed to evaluate the inflammatory response of the USC-laden scaffolds after implantation. The scaffolds loaded with USCs were implanted into a cranial defect model in rats to repair bone defects. Micro-computed tomography (μCT) analyses and histological evaluation were performed to evaluate the bone repair effects. Results GO modification enhanced the mechanical properties of the scaffolds. Scaffolds containing less than 0.5% GO had good biocompatibility and promoted USC proliferation and osteogenesis. The scaffolds loaded with USCs induced the M2-type differentiation and inhibited the M1-type differentiation of macrophages. The USC-laden scaffolds containing 0.1% GO exhibited the best capacity for promoting the M2-type differentiation of macrophages and accelerating bone regeneration and almost bridged the site of the rat cranial defects at 12 weeks after surgery. Conclusions This composite system has the capacity for immunomodulation and the promotion of bone regeneration and shows promising potential for clinical applications of USC-based, tissue-engineered bones.

Case Report: Osteomesh Cranioplasty in a 20-Year-Old Trauma Patient

In this study, we present a case of a 20-year-old male who suffered from severe traumatic brain injury with intracerebral hemorrhage, thus requiring decompressive craniectomy. Five months after, the patient underwent cranioplasty with the use of Osteomesh, a scaffold bone filler in reconstructing the post-operative cranial defect.

Biomimetic Three-layered Membranes Comprising (Poly)-ε-Caprolactone, Collagen and Mineralized Collagen for Guided Bone Regeneration

The distinct structural properties and osteogenic capacity are important aspects to be taken into account when developing guided bone regeneration membranes. Herein, inspired by the structure and function of natural periosteum, we designed and fabricated using electrospinning a fibrous membrane comprising (poly)-ε-caprolactone (PCL), collagen-I (Col) and mineralized Col (MC). The three-layer membranes, having PCL as the outer layer, PCL/Col as the middle layer and PCL/Col/MC in different ratios (5/2.5/2.5 (PCM-1); 3.3/3.3/3.3 (PCM-2); 4/4/4 (PCM-3) (%, w/w/w)) as the inner layer, were produced. The physiochemical properties of the different layers were investigated and a good integration between the layers was observed. The three-layered membranes showed tensile properties in the range of those of natural periosteum. Moreover, the membranes exhibited excellent water absorption capability without changes of the thickness. In vitro experiments showed that the inner layer of the membranes supported attachment, proliferation, ingrowth and osteogenic differentiation of human bone marrow-derived stromal cells. In particular cells cultured on PCM-2 exhibited a significantly higher expression of osteogenesis-related proteins. The three-layered membranes successfully supported new bone formation inside a critical-size cranial defect in rats, with PCM-3 being the most efficient. The membranes developed here are promising candidates for guided bone regeneration applications.

Poly-ε-Caprolactone/Fibrin-Alginate Scaffold: A New Pro-Angiogenic Composite Biomaterial for the Treatment of Bone Defects

We hypothesized that a composite of 3D porous melt-electrowritten poly-ɛ-caprolactone (PCL) coated throughout with a porous and slowly biodegradable fibrin/alginate (FA) matrix would accelerate bone repair due to its angiogenic potential. Scanning electron microscopy showed that the open pore structure of the FA matrix was maintained in the PCL/FA composites. Fourier transform infrared spectroscopy and differential scanning calorimetry showed complete coverage of the PCL fibres by FA, and the PCL/FA crystallinity was decreased compared with PCL. In vitro cell work with osteoprogenitor cells showed that they preferentially bound to the FA component and proliferated on all scaffolds over 28 days. A chorioallantoic membrane assay showed more blood vessel infiltration into FA and PCL/FA compared with PCL, and a significantly higher number of bifurcation points for PCL/FA compared with both FA and PCL. Implantation into a rat cranial defect model followed by microcomputed tomography, histology, and immunohistochemistry after 4- and 12-weeks post operation showed fast early bone formation at week 4, with significantly higher bone formation for FA and PCL/FA compared with PCL. However, this phenomenon was not extrapolated to week 12. Therefore, for long-term bone regeneration, tuning of FA degradation to ensure syncing with new bone formation is likely necessary.

The extract of concentrated growth factor enhances osteogenic activity of osteoblast through PI3K/AKT pathway and promotes bone regeneration in vivo

Background Concentrated growth factor (CGF) is a third-generation platelet concentrate product; the major source of growth factors in CGF is its extract; however, there are few studies on the overall effects of the extract of CGF (CGF-e). The aim of this study was to investigate the effect and mechanism of CGF-e on MC3T3-E1 cells in vitro and to explore the effect of combination of CGF-e and bone collagen (Bio-Oss Collagen, Geistlich, Switzerland) for bone formation in cranial defect model of rats in vivo. Methods The cell proliferation, ALP activity, mineral deposition, osteogenic-related gene, and protein expression were evaluated in vitro; the newly formed bone was evaluated by histological and immunohistochemical analysis through critical-sized cranial defect rat model in vivo. Results The cell proliferation, ALP activity, mineral deposition, osteogenic-related gene, and protein expression of CGF-e group were significantly increased compared with the control group. In addition, there was significantly more newly formed bone in the CGF-e + bone collagen group, compared to the blank control group and bone collagen only group. Conclusions CGF-e activated the PI3K/AKT signaling pathway to enhance osteogenic differentiation and mineralization of MC3T3-E1 cells and promoted the bone formation of rat cranial defect model.

Cranioplasty: Surgical Research

Background: Cranioplasty involves the repair of a cranial defect or deformation for cosmetic reasons, as well as long-term protection of the brain from the external environment. This work aims to evaluate and compare the efficacy, advantages and limitations of different materials used in cranioplasty. Methods: Prospective study of twenty-five patients who underwent cranioplasty for a skull bone defect by using different materials from March 2018 to March 2020. Results: The study included 13 males and 12 females. The defect was post-traumatic in 11 patient neoplastic in 13 patients and 1 patient was after decompressive craniectomy for malignant ischemia .When the defect was less than 80 cm² bone cement was used in 54.5%. When the defect was ≥ 80 cm² titanium mesh was used in 71.4 % of those cases. 72.0% of the patients (18 of 25) reported excellent cosmetic results, 24% (6 of 25) good, 4.0% (1 of 25) poor results. Conclusion: When the original bone flap is not available for cranioplasty titanium mesh is suitable for the large calverial bone defects. it is strong but hard to shape while bone cement is more suitable for small defects near the skull base as it is easy to shape but weak. Medpore and hydroxyapetite powder are better for pediatric defects as they don't hinder bone growth. Prefabricated bone flaps are effective but expensive and can't be used if cranioplasty is planned in the same operation.

Reconstruction of Craniectomy for Microvascular Decompression with Autologous Particulate Bone

Background and Study Objective Cranioplasty after microvascular decompression (MVD) is important for preventing postoperative complications such as headache. Autologous particulate bone is a common material for cranioplasty. The purpose of this study was to evaluate the effect of using autologous particulate bone to reconstruct the cranial defect produced by MVD. Patients and Methods Data were collected from January 2013 to December 2016 from 243 patients who underwent suboccipital retrosigmoidal craniectomy for MVD. The patients were then further divided into two groups: in the first group (from January 2013–October 2015), a cranioplasty was performed using a combination of bone dust (taken from a power drill) and particulate bone (harvested with a rongeur); in the second group (from November 2015–December 2016), the cranial defect was reconstructed using particulate bone alone. Healing of the cranial defect was observed during the follow-up. Results Early postoperative computed tomography (CT), performed during the hospital stay, revealed that the filling of the cranial defects of the first group was better than that of the second group. In addition, surgical-site infections (SSIs) occurred in 13 patients in the first group (9.92%) versus 2 patients in the second group (1.79%). The SSI rate of the first group was significantly higher than that of the second group (p < 0.05). Long-term follow-up CT demonstrated that the average reconstruction rate ((volume of the reconstruction area)/(volume of the cranial defect) × 100%) was 47.88% for the first group and 43.94% for the second group (p > 0.05). Conclusion The use of autologous particulate bone to reconstruct cranial defects after MVD has a good effect and is thus a useful and valuable technique. Bone dust may result in a higher incidence of SSI.

A Comprehensive 3D-Molded Bone Flap Protocol for Patient-Specific Cranioplasty

We present a detailed step-by-step approach for the low-cost production and surgical implantation of cranial prostheses, aimed at restoring aesthetics, cerebral protection, and facilitating neurological rehabilitation. This protocol uses combined scan computed tomography (CT) cross-sectional images, in DICOM format, along with a 3D printing (additive manufacturing) setup. The in-house developed software InVesalius®️ is an open-source tool for medical imaging manipulation. The protocol describes image acquisition (CT scanning) procedures, and image post-processing procedures such as image segmentation, surface/volume rendering, mesh generation of a 3D digital model of the cranial defect and the desired prostheses, and their preparation for use in 3D printers. Furthermore, the protocol describes a detailed powder bed fusion additive manufacturing process, known as Selective Laser Sintering (SLS), using Polyamide (PA12) as feedstock to produce a 3-piece customized printed set per patient. Each set consists of a “cranial defect printout” and a “testing prosthesis” to assemble parts for precision testing, and a cranial “prostheses mold” in 2 parts to allow for the intraoperative modeling of the final implant cast using the medical grade Poly(methyl methacrylate) (PMMA) in a time span of a few min. The entire 3D processing time, including modelling, design, production, post-processing and qualification, takes approximately 42 h. Modeling the PMMA flap with a critical thickness of 4 mm by means of Finite Element Method (FEM) assures mechanical and impact properties to be slightly weaker than the bone tissue around it, a safety design to prevent fracturing the skull after a possible subsequent episode of head injury. On a parallel track, the Protocol seeks to provide guidance in the context of equipment, manufacturing cost and troubleshooting. Customized 3D PMMA prostheses offers a reduced operating time, good biocompatibility, and great functional and aesthetic outcomes. Additionally, it offers greater than 15-fold cost advantage over the usage of other materials, including metallic parts produced by additive manufacturing.