Point-of-care treatment of geometrically complex midfacial critical-sized bone defects with 3D-Printed scaffolds and autologous stromal vascular fraction

Abstract

Critical-sized midfacial bone defects present a unique clinical challenge due to their complex three-dimensional shapes and intimate associations with sensory organs. To address this challenge, a point-of-care treatment strategy for functional, long-term regeneration of 2 cm full-thickness segmental defects in the zygomatic arches of Yucatan minipigs is evaluated. A digital workflow is used to 3D-print anatomically precise, porous, biodegradable scaffolds from clinical-grade poly-ε-caprolactone and decellularized bone composites. The autologous stromal vascular fraction of cells (SVF) is isolated from adipose tissue extracts and infused into the scaffolds that are implanted into the zygomatic ostectomies. Bone regeneration is assessed up to 52 weeks post-operatively in acellular (AC) and SVF groups (BV/DV = 0.64 ± 0.10 and 0.65 ± 0.10 respectively). In both treated groups, bone grows from the adjacent tissues and restores the native anatomy. Significantly higher torque is required to fracture the bone-scaffold interface in the SVF (7.11 ± 2.31 N m) compared to AC groups (2.83 ± 0.23 N m). Three-dimensional microcomputed tomography analysis reveals two distinct regenerative patterns: osteoconduction along the periphery of scaffolds to form dense lamellar bone and small islands of woven bone deposits growing along the struts in the scaffold interior. Overall, this study validates the efficacy of using 3D-printed bioactive scaffolds with autologous SVF to restore geometrically complex midfacial bone defects of clinically relevant sizes while also highlighting remaining challenges to be addressed prior to clinical translation.

Keywords: 3D printing; Critical-sized bone defects; Decellularized bone; Stromal vascular fraction; Yucatan pigs.

Fig. 1.. Point of care (POC) cell-based strategy for treating critical-sized midfacial bone defects.

CT images of each skull were used to custom-design porous PCL-DCB scaffolds which were 3D-printed with solid fixation tabs. At the time of surgery, custom-fit cutting guides were used to create 2 cm bilateral full-thickness defects within the zygomatic bone of Yucatan minipigs. Concurrently, subcutaneous adipose tissue was harvested from the dorsal lumbar region and the stromal vascular fraction of cells (SVF) was isolated and resuspended in fibrinogen. The cells were injected into the pore-spaces of the implanted scaffold without any pre-cultivation.

Fig. 2.. Design and Fabrication of custom PCL-DCB scaffolds.

(A) Pre-operative CT scans of the porcine skull were acquired and used as a template to design the custom PCL-DCB scaffolds. (i) Fixation tabs were added computationally to the scaffold body to complete the scaffold design. (ii) An in-house slicer program [17] was used to incorporate 800 μm isotropic pores into the scaffold and generate the g-code files for (iii) 3D-printing the PCL-DCB scaffolds. (B) Cone beam CT scans of each fabricated PCL-DCB scaffold were acquired. The central region of the scaffold (6 mm × 6 mm x 6 mm) was considered as representative volume and compared with the 3D theoretical design of the porous structure using a custom MATLAB^® program to quantitatively evaluate the percent agreement between the design and the printed parameters.

Fig. 3.. PCL-DCB scaffolds restored the native anatomy following critical-sized bone loss.

Representative images of zygomas harvested after 52 weeks of implantation at the zygomatic defect site exhibited restoration of native anatomy in both the AC and SVF experimental groups. In some cases, there appeared to be some exuberant bone growth at the dorso-caudal region of the implant (white circle). There was also significant soft-tissue coverage of the grafts. R: Rostral, C: Caudal; Scale bar = 2 cm.

Fig. 4.. Bone regeneration and functional osseointegration with the native bone.

(A) CT scans of the zygomatic defect sites depicting the region of interest (ROI, red), representative of the Empty, AC, and SVF groups at various post-operative timepoints. Note: Bony islands growing along the ventral aspect of the defect fill the defect volume and restore native anatomy (white arrow heads). Growth of bony extension from the caudal aspect of zygoma in a caudo-rostral fashion (red arrow heads). Orientation is conveyed by colored arrows: dorsal (red), ventral (yellow), rostral (blue) and caudal (green). Scale bar = 2 cm (B) Quantitative evaluation of bone volume/defect volume (BV/DV) within the ROI (n = 2 (E), 4 (AC) and 8 (SVF) defects). Note: Less bone was removed in the Empty group than originally planned, hence BV/DV is greater than zero even at the post-op time point. * indicates comparison with Empty group at the same time point whereas # indicates comparison with corresponding groups at PostOp. (C) Mechanical testing was performed to quantitatively assess osseointegration (n = 3 (AC) and 4 (SVF) samples) of the scaffolds with the native bone (* indicates p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5.. Analyzing the distribution of mineralized nodules within scaffolds.

(A) (i) 3D reconstructions of μCT scans of representative AC (defect volume = 4191.73 mm³) and SVF (defect volume = 4895.68 mm³) zygomas at 52 weeks with the scaffolds (red) segmented out and removed. (ii) Bone nodules formed within the pore spaces of the scaffold and bony extensions (asterisk) grew along the periphery. (iii, iv) With the bony extensions segmented out and removed, the sagittal and coronal views captured the spatial as well as volumetric distribution of new bone nodules within the scaffold pore spaces. The bone nodules were color coded based on their sizes, namely fine (<0.0216 mm³, grey), small (0.0216–0.108 mm³, red), medium (0.108–0.216 mm³, green), and large (>0.216 mm³, yellow). (B, C) Histograms quantitatively depicting the spatial bone distribution of all bone nodules within the scaffold in the AC and SVF groups (n = 2 and 4 for Acellular (AC) and SVF groups, respectively). Insets depict the spatial distribution of bone nodules, especially with volume larger than 0.0216 mm³, as their frequency is much lower as compared to those with volume less than 0.0216 mm³ (grey). C: Caudal, R: Rostral, M: Medial, L: Lateral. Scale bar = 1 cm (A i,ii); 0.5 cm (A iii,iv). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6.. Bone nodules closely associated with PCL-DCB struts.

(A) 3D rendered images of microCT scans of the defect site with the scaffold removed digitally (bottom panel) reveal that the bone nodules (yellow, top panel) nucleating within the pores of the scaffold are closely associated with the PCL-DCB struts (pink; asterisk). (B) Red dashed line indicates the level of histological section for Stevenel’s Blue (SB) staining. Scale bar = 2 cm (C) SB staining of representative samples (i, ii) elucidates presence of lamellar bone (black arrowheads) on the periphery of the scaffolds and woven bone (yellow arrows) within the scaffold pores. Scale bar = 4 mm (i, ii top panel); 100 μm (i, ii bottom panel) C: Caudal, R: Rostral, L: Lateral, M: Medial. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7.. Local inflammation within the PCL-DCB scaffolds.

(A) Representative Stevenel’s Blue staining of undecalcified samples (hard tissue histology) highlights the presence of diffuse fibrovascular stroma within the porous spaces and collagen around the exterior surfaces of the scaffold. Hemosiderin pigment was also detected within the scaffolds (black arrows). Scale bar = 4 mm (B) H&E staining of the harvested zygomas demonstrates the presence of lymphocytes (red arrow) within the PCL-DCB scaffold pore spaces and MNGCs (black arrow) in close association with the PCL-DCB struts (asterisk). Scale bar = 50 μm. (C) Semi-quantitative scoring of Stevenel’s Blue stained sections for assessing fibrosis, neovascularization, mineralization, hemorrhage and hemosiderin within the scaffolds. Scores indicate 0 = not present, 1 = minimal, 2 = mild, 3 = moderate and 4 = marked/severe. (D) H&E-stained sections were used to assess overall inflammation, and presence of polymorphonuclear cells, lymphocytes, macrophages and MNGCs within the scaffolds. Scores indicate: 0 = absent, 1 = rare, 2 = mild infiltrate, 3 = moderate/heavy infiltrate, 4 = packed infiltrate/sheets for MNGCs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8.. Presence of vasculature within the center of scaffold.

H&E staining of the whole cross section of representative decalcified Acellular (A) and SVF (A′) specimens with blue dashed line (insets) indicating the site of histological sectioning. Scale bar = 2 mm (Insets: 2 cm) (B-E, B′-E′) Zoomed in view of H&E stained specimens to highlight presence of vasculature (black arrowheads) within the center of the scaffold. Scale bar = 200 μm (B, B′); 20 μm (C-E, C′-E′). (F, F′) Immunostaining of decalcified specimens with DAPI, CD31 and α-smooth muscle actin (α-SMA) antibody highlights the presence of vasculature (white arrowheads) which is consistent with the observations from H&E stained specimens. * indicates PCL-DCB scaffold struts. Scale bar = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)