Mechanical engineering of hair follicle regeneration by in situ bioprinting

Highlights

• 3D bioprinting machine in situ bioprinting of Matrigel, Epi-SCs and SKPs directly into the wound site induced complete wound healing and functional tissue skin regeneration, the regenerated skin tissue including epidermis, dermis, blood vessels, hair follicles and sebaceous glands.

• 3D bioprinting machine in situ bioprinting could maintain the stemness of SKPs.

• 3D bioprinting machine in situ bioprinting only slightly decreased stem cell viability.

Abstract

Hair loss caused by various factors such as trauma, stress, and diseases hurts patient psychology and seriously affects patients' quality of life, but there is no effective method to control it. In situ bioprinting is a method for printing bioinks directly into defective sites according to the shape and characteristics of the defective tissue or organ to promote tissue or organ repair. In this study, we applied a 3D bioprinting machine in situ bioprinting of epidermal stem cells (Epi-SCs), skin-derived precursors (SKPs), and Matrigel into the wounds of nude mice to promote hair follicle regeneration based on their native microenvironment. The results showed successful regeneration of hair follicles and other skin appendages at 4 weeks after in situ bioprinting. Moreover, we confirmed that bioprinting only slightly decreased stem cell viability and maintained the stemness of the stem cells. These findings demonstrated a mechanical engineering method for hair follicle regeneration by in situ bioprinting which has potential in the clinic.

Introduction

Hair has many physiological functions, including protection against ultraviolet radiation, regulation of body temperature, sweat drainage, and tactile sensation [1]. Hair plays an important role in human physical appearance and hair loss caused by various factors such as trauma, stress, and diseases may not have an obvious effect on physical function, but can seriously affect social interaction [2], [3],which can negatively affect patient psychology, and quality of life. As of 2021, >2 billion people worldwide suffer from hair loss, with this impacting >250 million people in China. Currently, hair transplantation is the most effective method for treating hair loss [4]. The primary processes include hairline design, hair follicle (HF) extraction, HF separation, and transplantation into the scalp. Although the survival rate of HFs using these methods is very high, this approach only changes the distribution on their scalp without increasing their number. Moreover, despite hair transplantation becoming a well-established procedure for the treatment of hair loss, the lack of donor hair, coupled with the fact that hair loss is often progressive, remains a challenge in this field [5].

HFs are complex mini-organs that are formed during embryonic development through the interaction between epithelial cells and dermal papilla (DP) cells [6]. HFs are important skin appendage that progress through the cycles of anagen, catagen, and telogen [7], [8], which were previously thought to be formed solely during embryonic development. However, studies revealed that de novo HFs can develop from wounds under proper conditions [9]. Wound-induced HF neogenesis (WIHN) is a regenerative phenomenon that occurs widely in adult mammals [10] and might represent a potential treatment for hair loss through HF regeneration via wounding and Wnt/β-catenin pathway activation [11]. A previous study reported that during the early stages of wound repair, inflammatory factors production and migration into the wound area are associated with HF regeneration, and that after wound healing, decreases in the levels of these factors result in the disappearance of HF-regeneration signals [12]. Furthermore, challenges remain in the clinical application of these methods due to their limitations, such as induction of secondary damage.

Stem cell-based tissue engineering has emerged as the most thriving approach, aiming to reconstruct HFs in vitro to replace lost or damaged HFs as a consequence of disease, injury, or aging [13]. Previous studies have shown that DP cells from HFs have the ability to induce HF regeneration. Kageyama et al. [14] designed hair beads made of collagen and DPs were demonstrated to have the ability to promote HF regeneration in nude mice. However, due to the limitations of DP cells, such as the small amount, limited availability, and difficulty in maintaining their HF-inductive ability during in vitro culture, their widespread use is limited [15]. Skin-derived precursors (SKPs) are cells found in rodent and human skin, which when subcutaneously injected into mice, are incorporated into dermal papillae and induce HF regeneration. Furthermore, when transplanted in combination with Epi-SCs into excisional wounds in nude mice, SKPs induce de novo hair genesis [16], [17], [18]. These results suggest that SKPs have the potential to induce HF regeneration.

The development of three-dimensional (3D) bioprinting techniques has also expanded HF regeneration. Abaci et al. [19] designed a biomimetic approach for the generation of human HFs within human skin constructs by recapitulating the physiological 3D conformation of cells in the HF microenvironment using 3D-printed molds. Additionally, Kang et al. [20] 3D-bioprinted fibroblasts, human umbilical vein endothelial cells, DP cells, and epidermal cells in gelatin-alginate scaffolds and demonstrated that when the bioprinted scaffolds were transplanted into full-thickness wounds in nude mice, the HFs were regenerated. Although studies prepared scaffolds in vitro and implanted them in vivo, issues related to shape fitness wound likely be a problem for their clinical application. In situ bioprinting is an important branch of bio-printing, which describes the process of printing bioinks directly into the defective site according to the shape and characteristics of the defective tissue or organ to realize tissue or organ repair. This method could achieve accurate and rapid in situ bioprinting on the damaged site, and repair tissue based on its internal microenvironment. In this study, we used a 3D-bioprinting system for in situ bioprinting of Epi-SCs, SKPs, and matrigel into the wounds of nude mice (Fig. 1). The results demonstrated HF regeneration 4 weeks after in situ bioprinting and that the process of bioprinting only slightly decreased cell viability and did not affect the stemness of stem cells. This study provides a mechanical engineering method for HF regeneration by in situ bioprinting with considerable clinical applications.