4.1.3. Gelatin

Gelatin is a natural, biocompatible, non-immunogenic, hydrophilic, and biodegradable collagen derivative [153–155]. It is acquired via acid or alkaline hydrolysis of collagen into single molecules [156]. Being derived from a natural source, gelatin is characterized by having RGD cell-binding motifs that can enhance cellular attachment [157]; it can also promote cell proliferation and differentiation [158,159]. One of the major disadvantages of gelatin is its low mechanical properties [153,160], in addition to its thermal instability [161].

Gelatin crosslinking through the addition of chemical groups can help reduce these shortcomings [161]. Stiffness of gelatin hydrogel scaffold can be controlled through changing the ratio of crosslinking agents, such as methacryloyl, giving rise to gelatin– methacrylate (GelMA) [133,162,163], transglutaminase [134], or EDC [164], and through the incorporation of variable additives as starch [162] or polyethylene glycol diacrylate (PEGDA) [163]. Increased gelatin hydrogel scaffold degree of crosslinking and matrix stiffness were positively associated with increased osteogenic differentiation of MSCs [133,134,162–164]. Crosslinked gelatin hydrogel can also be modified to enhance osteogenic potential through incorporation of the bisphosphonate alendronate [163]. GelMA hydrogel with tunable stiffness was constructed by using different GelMA concentrations 10, 12, and 14 wt to yield hydrogel with stiffness 25.75 ± 1.21 kPa, 59.71 ± 8.87 kPa, and 117.82 ± 9.83 kPa, respectively. Osteogenic differentiation of PDLSCs, as well as expression of osteocalcin and Runx2, showed a significant increase with increasing matrix stiffness through activation of ERK1/2 signaling pathway [133].

Gelatin/starch-based hydrogel was fabricated with tunable stiffness from crosslinked gelatin with variable degrees of methacrylation (GelMA; 31%, 72%, and 95%) covalently bound to variable ratios of pentenoates modified starch (10 v% starch and 20 v% starch). Increasing the degree of methacrylation and combining crosslinked gelatin with starch, with subsequent increase in matrix stiffness, effectively promoted osteogenic differentiation of adipose stem cells (ASCs), as was evident by an increased ALP expression. GelMA 95% combined with starch showed the highest degree of osteogenic differentiation, while the highest degree of adipogenic differentiation was observed on the least crosslinked and most flexible gelatin hydrogel (GelMA 31%) [162].

Three-dimensional porous gelatin scaffolds crosslinked using EDC further demonstrated an increase in the elastic modulus from ~0.6 to ~2.5 kPa, without any change in the scaffold internal structure. BMMSCs cultured on EDC-crosslinked gelatin scaffolds with increased stiffness showed an increased osteogenic differentiation as evidenced by increased Runx2 and osteocalcin expression in vitro. Subcutaneous implantation of EDCcrosslinked gelatin scaffold loaded with BMMSCs transfected with adenovirus encoding BMP-2 in mice demonstrated an increased bone formation in vivo, as compared to the control, non-crosslinked scaffold with low stiffness [164].

Transglutaminase-crosslinked gelatin scaffold with variable stiffness was constructed, using gelatin concentrations of 3%, 6%, and 9%. The 9% gelatin gave rise to the highest stiffness (60.54 ± 10.45 kPa), while 3% gelatin resulted in the lowest stiffness (1.58 ± 0.42 kPa). BMMSCs encapsulated in the hydrogel with the highest stiffness demonstrated the highest osteogenic differentiation as revealed by ALP activity, calcified nodule formation, expression of SP7 transcription factor-2, Osx, Runx2, and osteocalcin [134].

Augmentation of GelMA with alendronate and PEGDA showed a positive effect on osteogenic differentiation of BMMSCs. GelMA and alendronate were added at different concentrations and grafted on gelatin molecules, followed by further crosslinking, using 20 wt.% PEGDA to improve hydrogel scaffold stiffness from 4 to 40 kPa. Osteogenic differentiation of grafted BMMSCs was promoted on stiffer hydrogel with higher alendronate concentration, as evident by upregulation of ALP activity, collagen type I, and osteocalcin expression, as well as calcium deposition [163].
