The Diamond Concept Enigma: Recent Trends of Its Implementation in Cross-linked Chitosan-Based Scaffolds for Bone Tissue Engineering

Abstract

An increasing number of publications over the past ten years have focused on the development of chitosan-based cross-linked scaffolds to regenerate bone tissue. The design of biomaterials for bone tissue engineering applications relies heavily on the ideals set forth by a polytherapy approach called the "Diamond Concept". This methodology takes into consideration the mechanical environment, scaffold properties, osteogenic and angiogenic potential of cells, and benefits of osteoinductive mediator encapsulation. The following review presents a comprehensive summarization of recent trends in chitosan-based cross-linked scaffold development within the scope of the Diamond Concept, particularly for nonload-bearing bone repair. A standardized methodology for material characterization, along with assessment of in vitro and in vivo potential for bone regeneration, is presented based on approaches in the literature, and future directions of the field are discussed.

Keywords: Bone Tissue Engineering; Chitosan-Based Scaffolds; Cross-linking; Diamond Concept; Growth Factors; Osteogenic Cells.

Figure 1

Comprehensive overview of the literature review presented in this manuscript. (A) “Diamond Concept” for Bone Regeneration, which encompasses an osteoconductive scaffold, osteogenic cells, osteoinductive mediators, and vascularization in a suitable mechanical environment. This builds the foundation for the organization of the review. (B) Increasing number of publications within the chitosan-based cross-linked scaffold/hydrogel domain for bone tissue engineering applications in the last 20 years. (C) The review’s novelty within the combined domain of chitosan based cross-linked hydrogel/scaffolds for bone tissue engineering applications implementing the Diamond Concept.

Figure 2

Results of literature search conducted for this manuscript. The search was performed using the keywords: Chitosan, Crosslinked (or Cross-linked), Bone, and Scaffold or Hydrogels. These results were also limited to publications from the last ten years and written in the English language. From this search, 188 articles were found to be suitable and screened further to examine the specific target of the study.

Figure 3

Material properties to consider following cross-linking of chitosan scaffolds. Each property is presented with common methodology for testing found in the literature.

Figure 4

Influence of scaffold design changes on the mechanical properties. (A) Compressive strength measurements of chitosan and collagen composite scaffolds at different ratios (75:25, 50:50, 25:75) prior to cross-linking. (B) Measurements of the same composite scaffolds after cross-linking with glyoxal. Both A and B were reproduced with permission from ref (106). Copyright 2021 Royal Society of Chemistry and the Centre National de la Recherche Scientifique. (C) Elastic modulus and compressive strength of gelatin chitosan composite scaffolds with increasing concentrations of bioglass (0 to 30%). C was reproduced and adapted under open access conditions (Creative Commons Attribution License) from ref (115). Copyright 2016 Kanchan Maji et al. and Hindawi.

Figure 5

Rheological property evaluation. (A) Effect of increasing chitosan-cysteine (CH–CY) solutions (1.5 to 2.5%) and difunctionalized PEG cross-linkers (0.3 to 0.6 w/v %) on G′ and G′′ values. Reproduced and adapted under open access conditions (Creative Commons Attribution License) from ref (100). Copyright 2022 Qing Min et al. and MDPI. (B) Percentages of published articles for inclusion of mechanical properties, rheological properties, both, and none.

Figure 6

Characterization of structural architecture for various modifications of chitosan scaffold. (A) SEM images of chitosan scaffolds with increasing concentrations of genipin (left −3.75%, right −7.5%). Reproduced and adapted under open access conditions (Creative Commons Attribution License) from ref (72). Copyright 2017 Simona Dimida et al. and Hindawi. (B) Total porosity measurements (T.Po), structure thickness (St.Th), and specific surface (Sp.S) for chitosan scaffolds with increasing graphene oxide (GO) percentages (0, 0.5, 3%). Reproduced and adapted under open access conditions (Creative Commons Attribution License) from ref (177). Copyright 2019 Sorina Dinescu et al. and MDPI. (C) Percentage of porosity for sodium alginate and chitosan composite scaffolds with and without the addition of collagen and graphene oxide using liquid displacement method. Reproduced and adapted with permission from ref (138). Copyright 2018 American Chemical Society.

Figure 7

Degradation profiles of composite chitosan scaffolds in lysozyme. (A) Chitosan (CS) and polycaprolactone (PCL) scaffolds with different concentrations of polymers (100:0, 20:80, 40:60, 0:100) cross-linked with genipin. Reproduced and adapted with permission from ref (119). Copyright 2022 Elsevier. (B) Chitosan/hydroxyapatite/collagen composite (Ha-Col1-CS) scaffolds before and after cross-linking with different methodologies: (1) DHT – dehydro-thermal treatment, (2) IR – irradiation, (3) GTA – glutaraldehyde, and (4) HEMA – 2-hydroxyethyl methacrylate. Reproduced and adapted under open access conditions (Creative Commons Attribution License) from ref (111). Copyright 2019 Md. Shaifur Rahman et al. and Springer.

Figure 8

Common methodologies for in vitro studies using cell cultures in the literature. These steps include testing for biocompatibility, osteogenic differentiation, and biomineralization.

Figure 9

Assessment of (A) osteoblastic differentiation (ALP activity) and (B) mineralization potential (Alizarin Red Staining) from MG-63 cells over 21 days for chitosan scaffolds with and without tripolyphosphate cross-linker. Reproduced and adapted with permission from ref (98). Copyright 2012 Wiley Periodicals, Inc.

Figure 10

Common osteoinductive mediators in the literature for chitosan-based cross-linked scaffolds.

Figure 11

General mechanisms that mediate the release of factors from scaffolds and common methodologies used to assess this release.

Figure 12

Cumulative release profiles of different encapsulants from scaffolds. (A) Release percentage of pentoxifylline at the 6 h time point for chitosan scaffolds cross-linked with genipin, pectin, or freeze-dried. Reproduced and adapted with permission from ref (73). Copyright 2015 IOP Publishing. (B) Cumulative release of rhBMP2 from PLGA microspheres, which will be placed in composite chitosan scaffolds with mineralized collagen I. Reproduced and adapted with permission from ref (135). Copyright 2017 Wiley Periodicals, Inc. (C) Release patterns of rhBMP-2 and vancomycin (VAN) from methacrylated chitosan photo-cross-linked scaffolds with or without PLGA microspheres. Reproduced and adapted with permission from ref (169). Copyright 2021 Elsevier. (D) Cumulative release percentage of tetracycline hydrochloride from carboxymethyl chitosan/oxidized gellan gum gels with hydroxyapatite (HAp/gel) and gels with magnetic hydroxyapatite/gelatin microspheres (MHGMs). Reproduced and adapted with permission from ref (103). Copyright 2022 Elsevier.

Figure 13

Effect of sphingosine 1-phosphate (S1P) loaded silica nanoparticles (MSNs) addition to N,O-carboxymethyl chitosan (NOCC) and aldehyde hyaluronic acid (aHa) nanocomposite hydrogels on capillary network formation in vitro and in vivo. (A, B) Results of CAM assay for examination of angiogenesis and quantification of vascular area. (C, D) HE staining images at the 2-week time point after implantation into a mouse. Arrows present the blood vessels. D presents the counted number of vessels shown. Reproduced and adapted with permission from ref (105). Copyright 2022 Royal Society of Chemistry.

Figure 14

In vivo studies using (A–C) MicroCT and (D) HE staining. (A) Images of mouse tibial fractures treated with nothing (SHAM) or chitosan scaffolds with hydroxyapatite (HA) and pyrophosphatase (P). Reproduced and adapted under open access conditions (Creative Commons Attribution License) from ref (88). Copyright 2020 Kaushar Jahan et al. and Springer Nature. (B, C) Imaging and quantitative analysis of a rat cranial defect model at 12 weeks postsurgery treated with control, chitosan and PCL composite scaffolds, and composite scaffolds with Alendronate. (C) Quantification of the mature bone area, bone volume/tissue volume, and bone mineral density for these samples. Both B and C were reproduced and adapted with permission from ref (119). Copyright 2022 Elsevier. (D) Staining for new bone formation in defects treated with nothing (top), chitosan scaffold (middle), or a chitosan scaffold loaded with copper (bottom). Within this figure, the new bone (NB) and old bone (OB) are distinguished. Reproduced and adapted with permission from ref (99). Copyright 2014 Wiley Periodicals, Inc.

Figure 15

Common methodologies for in vivo studies using animal models in the literature. These are divided into 3 components: imaging studies, histological staining, and quantitative assays.