The field of tissue engineering aims to develop viable substitutes with the ability to repair and regenerate the functions of damaged tissue. Common practices to supplement bone regeneration in larger defects include bone graft biomaterials such as autografts, allografts, xenografts, and synthetic biomaterials. Autologous bone grafting is the current gold-standard procedure used to replace missing or damaged bone. However, these grafts have disadvantages such as donor site morbidity, limited availability, and the need for a secondary surgery. The focus of this study is to tissue engineer a lyophilized gelatin composite sponge composed of hydroxyapatite (HA), chitin whiskers (CW), and preparations rich in growth factors (PRGF) to provide sufficient structural support to the defect site while enhancing the body’s own reparative capacity, ultimately eliminating the need for autologous tissue harvesting or repeat operations. The present study investigates several in vitro evaluations on multiple compositions of modified gelatin sponge scaffolds for use in bone graft applications. Gelatin sponges were fabricated via freeze-drying, enhanced with PRGF, HA, and/or CW, and cross-linked with 50 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) either during or post gelation. Initial evaluation of all scaffold combinations indicated that scaffolds released contents up to 90 days, EDC cross-linking during gelation allowed for more protein release, and had the ability to swell. Since the incorporation of PRGF, HA, and CW increased cell infiltration, and production of cell-created mineral matrix over 90 days in culture, these scaffolds were further characterized. Freeze-dried gelatin sponges enhanced with PRGF, HA, and CW and cross-linked during gelation with EDC (PHCE) were mineralized (M) in 5x revised simulated body fluid (r-SBF) for 1 hour to create a bone-like mineral surface. Gelatin EDC scaffold controls (GE), GE-M, PHCE, and PHCE-M scaffolds were characterized for their ability to swell, mineralizing potential, surface morphology, growth factor incorporation and release, uniaxial compression properties, and cell attachment, proliferation, infiltration, and protein/cytokine secretion.. After mineralization, scanning electron microscopy showed sparse clusters of mineral deposition for GE-M scaffolds while PHCE-M scaffolds exhibited a more uniform mineral deposition. Both GE and PHCE scaffolds were porous structures that swelled up to 50% of their original volume upon hydration. Over 21 days incubation, PHCE-M scaffolds cumulatively released about 30% of their original protein content, significantly more than all other scaffolds. Multiplex Luminex assays confirmed the successful incorporation of PRGF growth factors within PRGF sponges. For acellular uniaxial compression testing, PHCE-M scaffolds reported lower Young’s modulus values (1.3 - 1.6 MPa) when compared to GE and GE-M scaffolds (1.6 – 3.2 MPa). These low modulus values were comparable to values of tissue found in early stages of bone healing. DAPI (4',6-diamidino-2-phenylindole) staining and imaging showed an increase in initial cell attachment and infiltration of PHCE and PHCE-M scaffolds on day 1. GE-M scaffolds also appeared to attach more cells than the GE control. MTS cell proliferation assay results indicated that on days 4 and 7, PHCE scaffolds increased cell proliferation (compared to GE controls). MTS also illustrated that the addition of a mineral coating increases and decreases cell proliferation on GE-M and PHCE-M scaffolds, respectively. Multiplexer analysis of MG-63 protein/cytokine secretion suggests that cells are responding in a bone regenerative fashion on all scaffolds, as evidence of osteocalcin secretion. Little to no secretion of osteopontin, IL-1β, and TNF-α demonstrates that scaffolds are not influencing cells to secrete factors associated with bone resorption. The compressive mechanical properties of cellularized scaffolds did not differ much from acellular scaffolds. The collective results indicated increased cellular attachment, infiltration, and bone regenerative protein/cytokine secretion by cells on GE-M scaffolds, which support the addition of a bone-like mineral surface on GE scaffolds. Cellularized PHCE and PHCE-M scaffolds report similar advantages as well as Young’s modulus values in the range of native tissues present in the early stages of bone healing. The results of this study propose that the developed PHCE and PHCE-M scaffolds exhibit good cellular responses and mechanical properties for use in early bone healing applications.

Composites, in which the matrix and the reinforcing fillers are respectively, poly(acrylic acid) (PAA) with two different molecular weights, and chitin whiskers (CHW) were successfully prepared using an evaporation method. The weight fraction of CHW was varied from 0.03 to 0.73. Mechanical and thermal properties and crystallinity of the composites were characterised using tensile testing, differential scanning calorimetry, thermogravimetric analysis and X-ray diffraction. The tensile strength of the composite increased up to 11 wt % CHW after which it decreased. XRD characterisation showed a decrease in crystalline index, crystalline size, chitin crystalline peak and intensity as the content of PAA and its molecular weight increased. Raman spectroscopy was used for the first time to monitor the deformation of chitin film and CHW reinforced PAA composites. The Raman band located at 1622 cm^(-1) was monitored for deformation. On application of tensile deformation the Raman band initially located at 1622 cm^(-1) shifted toward a lower wavenumber. Raman band shift rates of -1.85 cm^(-1)/% for chitin film and -0.59 and -0.25 cm^(-1)/% for 73 and 23 wt % CHW content, respectively, were measured. The modulus of a single chitin whisker and composites were found to be 115, 37 and 16 GPa respectively, for a two dimensional (2D) in-plane distribution of CHW. CHW within a PAA matrix did not show any preferential alignment in a polarised Raman. The Raman intensity ratio〖 I〗_1698 /I_1622 showed that the strongest interaction of the carboxylic group in the composites occured at 3 wt % CHW content. The interaction gradually reduced as the CHW content increased. 〖 CaCO〗_3 crystals were grown in CHW, PAA and CHW/PAA composites by a solution and evaporation casting method. In the absence of PAA and CHW, rhombohedral calcites were observed while rod-like aragonite polymorphs were seen when only PAA was used as a template. In the presence of only CHW, a morphological mixture of ellipsoidal and disc shape with traces of rhombohedral aggregate calcite were the features. In the presence of both PAA and CHW, the rhombohedral shape showed roughness with irregular faces while the vaterite polymorph continued to agglomerate with the observation of porosity at higher CHW content. The vaterite particles gradually decreased as the CHW content was decreased. At lower CHW content aragonite polymorph growth was favoured to the detriment of calcite. The results showed that the vaterite polymorph can be grown even at higher filler loading. The effect of 〖 CaCO〗_3 growth on the mechanical properties of CHW reinforced PAA indicated that better mechanical properties can be achieved at a CHW content of 3 wt % when compared with neat PAA and when 〖 CaCO〗_3 was not incorporated into the CHW/25PAA composites.