In situ photochemical crosslinking-assisted collagen compression process toward dense collagenous construct mimicking biophysical and biochemical cell environments for promoted wound repair

Abstract

The present thesis is about the development of the stable, reproducible, and stiffness tunable collagen compression system and process to accomplish the biomimetic dense collagenous construct for wound repair. Regarding that various biophysical and biochemical cell environments of the native ECM, such as stiffness, topography, and biochemical components, are crucial factors to regulate the cellular activities in the damaged tissue repair, many studies have paid substantial interest in the development of the tissue-engineered construct recapitulating the cell environments. Accordingly, a number of collagen hydrogel-based biofabrication processes have been actively developed to utilize the structural component of the native ECM and its in vivo-like fibrous structure. Among various biofabrication processes, the collagen compression process has great potential in realizing the more in vivo-like biophysical cell environments, given that it can simply produce the dense collagen fibrillar structure by the physical compaction of the collagen hydrogel and thus improve the stiffness ~ 0.1–1 MPa. Despite the great benefits, the clinical application of the fabricated construct was hardly achieved due to the two issues in the fabrication system and process: (1) Instability and low reproducibility in the process by the manual compression process and (2) restriction in the improvement of mechanical properties for surgical transplantation. In addition to that, the development of the collagenous construct that mimics complex biophysical and biochemical cell environments, such as the tissue stiffness on the order of ~ 10 MPa and tissue-specific biochemical composition, is still challenging issue, requiring a novel collagen-based biofabrication process to accomplish it. In this thesis, the robust collagen compression system and in situ photo-chemical crosslinking-assisted collagen compression (IPC-CC) process were developed to overcome the limitations in the conventional approach. The robustness of the collagen compression system was primarily established for the stability and reproducibility in the fabrication process, with consideration of the material characteristics of the collagen hydrogel, i.e. poroelastic/biphasic material characteristics. With the investigations on its theoretical model and parametric studies, we demonstrated that the compression rate was closely related to the occurrence of the internal stress, i.e. pore pressure and thus lead to the structural breakage of the collagen fibrillar structure. The configuration of the robust collagen compression system and controller system was designed to maintain a desired compression rate until reaching the final compressive pressure, to prevent the undesirable pore pressure increase. Thus, the robust collagen compression system could reveal its stability and reproducibility in the collagen compression process, producing the structurally stable compressed collagen without significant deformation and breakage, even the final compressive pressure of ~ 190 kPa. Based on the establishment of the stability and reproducibility in the collagen compression process, we developed the IPC-CC process to improve the mechanical properties of the compressed collagen for the in vivo-like biophysical environment and stability in surgical transplantation. While the riboflavin/UV mediated photochemical crosslinking process has been widely utilized to increase the stiffness of the compressed collagen with the little toxicity, our parametric studies found that the stiffness increase by the photochemical crosslinking was restricted by the rehydration of the compressed collagen in the riboflavin solution. The rehydration would lower the efficiency of the photochemical crosslinking by the reduced reaction sites with the relatively sparse distribution of the collagen fibrillar structure. In this regard, we integrated the collagen compression process and photochemical crosslinking process to achieve the simplicity in process and chiefly the enhancement in crosslinking efficiency by maintaining the densely packed fibrillar structure for the higher reaction sites. Hence, the IPC-CC process could produce the dense collagenous construct having a mechanical stiffness on the order of ~ 10 MPa. In addition, the IPC-CC process could be advanced to the collagen micro-patterning process, i.e. in situ photochemical crosslinking-assisted collagen micro-patterning (IPC-CμP) process. The IPC-CμP process could generate the micro-patterns on the order of ~ 1 μm, demonstrating its potential in recapitulating in vivo micro-topography on the collagenous construct. Utilizing the robust collagen compression system and IPC-CC and IPC-CμP processes, two types of the dense collagenous constructs mimicking the biophysical cell environment were developed for the cornea or skin wound repair: (1) dense collagenous biocomposite for limbal epithelial stem cell (LESC) carrier and (2) dense collagenous dermal substitute for skin epithelial wound repair. The dense collagenous biocomposite could be obtained based on the robust collagen compression system by adopting the decellularized corneal tissue, known as decellularized corneal lenticule (dCL), which maintained the mechanical stiffness of the native tissue. Thus, the biophysical cell environment of the native cornea and stability in surgical transplantation could be readily achieved by incorporating the dCL inside the compressed collagen. The clinical efficacy of the biocomposite was verified by the in vivo limbal stem cell deficiency model. The in vitro LESC culture and in vivo transplantation/regeneration proved that the biocomposite effectively supported the native LESC functions and promoted the reconstruction of the damaged limbal tissue. While the biocomposite had facileness in obtaining the in vivo-like biophysical environment, its general use is would be restricted by the low availability of the dCL driven from the human cornea. The second approach based on the IPC-CC and IPC-CμP processes had simplicity and versatility in the fabrication process as well as the high availability of the raw material. Thus, the dense collagenous dermal substitute was developed utilizing the IPC-CC and IPC-CμP processes for the clinical demonstration. More complex biophysical cell environments of the native skin, including stiffness of ~ 30 MPa and micro-topography of in vivo-like wound gap, could be realized on the dense collagenous construct. The suture retention test found that the improved mechanical properties also provided the stability in surgical transplantation. The in vitro skin epithelial wound healing assay with the newly developed platform revealed the potential in the clinical application, showing that the skin epithelial wound repair could be promoted by the developed dense collagenous dermal substitute. We finally developed the dense collagenous construct mimicking complex biophysical and biochemical cell environments by adopting the corneal-derived decellularized extracellular matrix (Co-dECM) to the developed collagen compression system and process. Considering that the Co-dECM contains sufficient tissue-specific components, the application of Co-dECM to the collagen compression process would produce the dense collagenous construct mimicking biochemical cell environment. On the other hand, our study found that the Co-dECM hydrogel was incompatible with the collagen compression process due to its limited fibrillar interconnectivity. Thus, we suggested a method to increase the fibrillar interconnectivity by intermixing the Co-dECM with the conventional collagen for the collagen compression process. With the parametric studies on the Co-dECM intermixing ratio, the optimal intermixing ratio could be found to obtain the structural stability and uniformity of the fabricated construct, while maintaining the more than 2-fold higher glycosaminoglycan (GAG) content in the compressed collagen. The in vitro corneal keratocyte and epithelial cell culture revealed that biochemical components of the Co-dECM incorporated in the compressed collagen effectively promoted the native corneal cell functions, such as quiescent keratocyte phenotype and epithelial proliferation. However, the mechanical tensile test found that the Co-dECM intermixing could reduce the mechanical properties of the compressed collagen, indicating that additional mechanical improvement was essential for the biophysical cell environment and its clinical application. Lastly, the Co-dECM intermixing method was applied to the IPC-CC process to develop a cornea-mimetic dense collagenous construct that mimics both the biophysical and biochemical cell environments of the native cornea. The IPCC-process and Co-dECM intermixing method successfully produced the cornea-mimetic dense collagenous construct having a collagen density ~ 200 mg/ml, elastic modulus of ~ 12–15 MPa, and ultimate tensile strength of ~ 2–4 MPa, sufficient for clinical application. The GAG content also maintained at a higher level than other collagenous constructs without Co-dECM intermixing. The clinical efficacy of the cornea-mimetic dense collagenous construct was evaluated by the in vivo corneal perforation model that resulted in various complications like corneal scarring, bacterial infection, leakage of aqueous humor, and iris synechiae. The in vivo test found that the cornea-mimetic dense collagenous construct could at by the surgical suture, protect the eye from leakage of aqueous humor, and promote the normal repair of the corneal perforation, while the untreated model resulted in the iris synechiae by the leakage of the aqueous humor. In conclusion, the robust collagen compression system and IPC-CC process proposed in this thesis are expected to provide a versatile and powerful tool to develop the biomimetic dense collagenous construct environments of the various native tissues, thereby broadening its applications for promoted wound repair. Furthermore, the method of dECM intermixing for the biochemical cell environment also could amplify the clinical efficacy of the dense collagenous construct by allowing the recapitulation of more complex cell environments with the presented system and process. These advancements are also could be expanded to various applications in the fields of tissue-engineering and biomedical research, such as the development of the physiologically relevant in vitro organ model.