Fabrication of biocompatible porous scaffolds based on hydroxyapatite/collagen/chitosan composite for restoration of defected maxillofacial mandible bone

Abstract

Fabrication of scaffolds from biomaterials for restoration of defected mandible bone has attained increased attention due to limited accessibility of natural bone for grafting. Hydroxyapatite (Ha), collagen type 1 (Col1) and chitosan (Cs) are widely used biomaterials which could be fabricated as a scaffold to overcome the paucity of bone substitutes. Here, rabbit Col1, shrimp Cs and bovine Ha were extracted and characterized with respect to physicochemical properties. Following the biocompatibility, degradability and cytotoxicity tests for Ha, Col1 and Cs a hydroxyapatite/collagen/chitosan (Ha·Col1·Cs) scaffold was fabricated using thermally induced phase separation technique. This scaffold was cross-linked with (1) either glutaraldehyde (GTA), (2) de-hydrothermal treatment (DTH), (3) irradiation (IR) and (4) 2-hydroxyethyl methacrylate (HEMA), resulting in four independent types (Ha·Col1·Cs-GTA, Ha·Col1·Cs-IR, Ha·Col1·Cs-DTH and Ha·Col1·Cs-HEMA). The developed composite scaffolds were porous with 3D interconnected fiber microstructure. However, Ha·Col1·Cs-IR and Ha·Col1·Cs-GTA showed better hydrophilicity and biodegradability. All four scaffolds showed desirable blood biocompatibility without cytotoxicity for brine shrimp. In vitro studies in the presence of human amniotic fluid-derived mesenchymal stem cells revealed that Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT scaffolds were non-cytotoxic and compatible for cell attachment, growth and mineralization. Further, grafting of Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT was performed in a surgically created non-load-bearing rabbit maxillofacial mandible defect model. Histological and radiological observations indicated the restoration of defected bone. Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT could be used as an alternative treatment in bone defects and may contribute to further development of scaffolds for bone tissue engineering.

Keywords: AF-MSCs; Biocompatibility; Bone tissue engineering; Chitosan; Collagen; Hydroxyapatite; Mandible bone defect; Scaffold.

Fig. 1

Schematic representation for the fabrication of porous Ha·Col1·Cs scaffold. (A1–A5) Isolation steps of Col1. (B1–B3) Extraction and processing of Ha. (B4) Dispersion of HA powders in the water. (C1) Extracted brine shrimp derived Cs. (D1) Mixing of Cs in the Col1 solution at 2:1 ratio. (B4 and D2) Mixture of Ha slurry, and Cs-Col1 solution at 60:40 ratio and homogenization. (E) Freeze-dried Ha·Col1·Cs scaffold without cross-linkers. Cross-linked fabricated scaffold with physical method (DHT and IR) (F1-F2) and chemical method (HEMA and GTA) (G1-G2)

Fig. 2

FTIR spectra of Ha·Col1·Cs scaffolds with and without cross linkers. a Ha·Col1·Cs (without cross-linkers) spectra with corresponding individual constituents namely Ha, Cs and Col1. b FTIR analysis for Ha·Col1·Cs-DHT, Ha·Col1·Cs-HEMA, and Ha·Col1·Cs-GTA where Ha·Col1·Cs (without cross-linkers) served as reference. c Effects of various radiation doses on Ha·Col1·Cs scaffold as a cross-linker. d Comparative analysis of FTIR spectra between Ha·Col1·Cs (without cross-linkers), Ha·Col1·Cs-DHT, Ha·Col1·Cs-IR (25 kGy), Ha·Col1·Cs-HEMA and Ha·Col1·Cs-GTA

Fig. 3

SEM micrographs of different Ha·Col1·Cs scaffolds and human bone graft (HBG) from horizontal cross-sections at the middle region of the scaffold. a Ha·Col1·Cs scaffold without cross-linked. b Ha·Col1·Cs-IR scaffold cross-linked with 25 kGy gamma irradiation. c Ha·Col1·Cs-DHT scaffold cross-linked with DHT method. d Ha·Col1·Cs-GTA scaffold cross-linked with GTA solution. e Ha·Col1·Cs-HEMA scaffold cross-linked with HEMA. f Human bone graft served as positive control

Fig. 4

Physicochemical characterization of the fabricated scaffolds. a Porosity range of the scaffolds. b Density of the fabricated scaffolds. c Swelling percentage of Ha·Col1·Cs (non-cross-linked and cross-linked) scaffolds at different soaking time: (left) swelling percentage of scaffold composition on the overall water uptake and (right) swelling percentage of scaffold material itself. d Enzymatic degradation studies of Ha·Col1·Cs (non-cross-linked and cross-linked) scaffolds. e Mechanical strength. f Stabilities of Ha·Col1·Cs scaffolds (non-cross-linked and cross-linked) in aqueous solution: (left) stability test at pH 4.0 and (right) stability test at pH 7.0

Fig. 5

In vitro cytotoxicity and human blood biocompatibility of Ha·Col1·Cs scaffolds and its constituents. a Brine shrimp lethality assay. b RBC hemolysis biocompatibility assay. PC positive control, and distilled water served as NC negative control

Fig. 6

AF-MSC attachment, growth and mineralization analysis in presence of various Ha·Col1·Cs scaffolds. a AF-MSC attachment and growth in presence of the formulated scaffolds powder. b In vitro mineralization of AF-MSCs in presence of distinct scaffolds. Calcification was evidenced by Alizarin Red (ARS) staining

Fig. 7

In vivo grafting of scaffold in the surgically created rabbit maxillofacial mandible defect (non-load bearing) model. a Surgical and implantation procedures. (A1) Surgical incision showed the site of mandible to be drilled. (A2) Drilled defect chamber in the mandible of rabbits. (A3) Defect filled with scaffold. (A4) Sutured incision. b Post-grafting recovery observation from day 7 to day 28

Fig. 8

Post-operative histological and radiological analysis. a Histological analyses of the treated defects after 4 months. Defected mandible without any implant/graft (first lane), Ha·Col1·Cs-IR (second lane), and Ha·Col1·Cs-DHT (third lane) whereas native bone (NB) and newly bone growth (NBG) are distinguished. b Representative radiological images during the recovery period in the Ha·Col1·Cs-IR group from (B1) day 1 to (B4) 4 months