2.1 Introduction to stem cells

Since the 20th century, stem cell and regenerative medicine technology have been one of the hot frontiers in the international biomedical field, which plays an irreplaceable role in safeguarding human life and health in improving the quality of human survival and extending human life expectancy.⁵⁰ Stem cells are undifferentiated cells characterized by a high capacity for proliferation and self-renewal as well as clonality usually derived from a single cell and differentiated into different types of cells and tissues, these properties may vary between stem cells.⁵¹ For example, embryonic stem cells (ESCs) obtained from blastocysts and adult humans have different properties, with ESCs from blastocysts having higher tissue specificity. According to the stage of stem cell development, ESCs and adult stem cells (ASCs) are classified.⁵² ESCs can self-regenerate and differentiate into all tissues in vivo. Numerous animal studies have shown that ESCs promote neural regeneration in neuron-deficient and chronically denervated rat models.⁵³ However, the application of ESCs is currently limited due to ethical issues. In addition to this, adult stem cells include cells present in tissues or organs that have the potential for further differentiation, the ability to self-renew, and the ability to differentiate into the major types of specialized cells of the tissue from which they originate. ASCs mainly include mesenchymal stem cells (MSCs), myogenic stem cells (MDSCs), neural stem cells (NSCs), and urogenic stem cells (USCs).⁵⁴

MSCs are one kind of adult stem cells that originate from the early mesoderm and self-renewing mesodermal cells with multidirectional differentiation potential, which can be isolated from adult bone marrow, dental pulp, adipose tissue, umbilical cord, and other tissues.⁵⁵ Among the stem cell family, MSCs are the most widely studied and used stem cells in the field of regenerative medicine. According to different sources, they can be divided into: adipose-derived MSCs, umbilical cord MSCs, bone marrow MSCs, and so on.⁵⁶ There are relevant research tables showing that it has good application prospects in the field of regenerative medicine, and some MSCs have already entered clinical application.⁵⁷ MSCs have many advantages such as easy isolation, high in vitro expansion, low immunogenicity, and targeted differentiation into neural tissue cells.⁵⁷ MDSCs have long-lasting self-renewal and multigerm differentiation ability and can differentiate not only into mesodermal myoblasts, osteoblasts, chondrocytes, adipocytes, endothelial cells, hematopoietic cells, but also into ectodermal neuronal cells.⁵⁸ With immunity, donor cells can still be detected weeks after the acute inflammatory response and the damaged nerve can still be functionally regenerated after transplantation of human MDSCs.⁵⁹ NSCs transplantation provides a new therapeutic idea for neurological injury diseases and promotes the development of neural regeneration. NSCs not only have the function of cell replacement, but also play the function of immunomodulation, and regulate the activation of complement.⁶⁰ Dental stem cells (DSCs) are a population of embryonic neural crest-derived adult stem cells that are homologous to neural tissue.⁶¹ DSC has stronger proliferation and multidirectional differentiation ability than MSC from other tissues and is one of the ideal stem cell sources for tissue regeneration because it is easy to obtain with features of less invasive and less immune rejection.⁶² Urine-derived stem cells are new stem cells with good expansion capacity, multilineage differentiation potential, and paracrine function.⁶³ Increasing interests in this aspect shows that it can be isolated from urine samples with the advantages of easy collection, noninvasive sampling, and few ethical issues.⁶⁴

Stem cell therapy represents an important trend in the development of medicine for regeneration. The role of stem cell therapy in repairing, restoring or reconstructing human tissue and organ damages is becoming more and more prominent. The application of stem cells and their derivatives has solved many medical problems in recent years. Skin and bone injuries often lead to severe disability, and exploration of promising therapeutic strategies is of great importance. As the largest organ in the body, the skin is vulnerable to injury and weak in self-repair. Furthermore, the bones are vulnerable to injury causing greater damage, relying solely on self-repair cycles that leading to more pain on patients. These reasons making skin and bone a current research hotspot in regenerative medicine.^47-49

2.2 Stem cells promote skin tissue regeneration

MSCs were first observed in the bone marrow in 1867 by Kornheim, who found that these cells may be the source of fibroblasts involved in wound repair.⁶⁵ Studies have confirmed that stem cells play an important role in the treatment of various skin injuries⁶⁶ and numerous scholars have studied their regenerative repair mechanism.^{67, 68} Additionally, stem cells can effectively suppress the inflammatory response and assist in wound repair for skin regeneration through their immunomodulatory effects (Table 1). In this article, we discuss the use of stem cells in several stages of wound healing.

The inflammatory phase plays a crucial role in the wound healing process by inducing the recruitment of immune cells to prevent continued pathogenic damage to the organism.^{69, 70} During the hemostatic phase, stem cells migrate to the local wound site and induce vasoconstriction and platelet agglutination to promote blood clotting.⁷¹ To produce hemostatic and anti-inflammatory effects, a closed wound is temporarily formed on the wound surface.⁷² A few hours after wound formation, the inflammatory phase begins, which is characterized by local congestion and plasma exudation. Some studies have used flow cytometry to examine cell proliferation and demonstrated that MSCs have potent immunomodulatory effects and possess antimicrobial properties by regulating the functional properties of T cells, B cells, and dendritic cells.⁷³ In the early stages of inflammation, the level of wound inflammatory factors increases, including the anti-inflammatory factors interleukin (IL)-4, IL-10, IL-13, and others. In an experiment on rats, MSCs increased the level of anti-inflammatory factors, whereas that of proinflammatory factors and tumor necrosis factor (TNF)-α, gamma-interferon, IL-1, IL-6, IL-8, intercellular adhesion molecule-1, and others was decreased.⁷⁴

Macrophages play a major immunomodulatory role after 3 days of wound formation and are the key regulators of the local inflammatory microenvironment. After injecting the wound surface of diabetic mice with MSCs, it was found that macrophages were recruited to the wound in large numbers, which in turn induced macrophages to acquire the M2 anti-inflammatory phenotype.⁷⁵ This caused the wound to produce an immune response that inhibited bacterial and other undesirable proliferation and promoted tissue regeneration. Another reported further studies that by experimenting with mouse bone marrow MSCs. Assessment of the immunosuppressive effects of different immune cells. MCSs promote macrophage polarization while inhibiting B-cell differentiation by regulating IL-1 receptor antagonists.⁷⁶ The anti-inflammatory effects of MSCs have been well illustrated. Furthermore, MSCs induce the production of IL-10 by macrophages at inflammatory sites. IL-10 is a cytokine with anti-inflammatory properties and induces regeneration of traumatized tissue, which greatly contributes to wound healing and lays the foundation for wound repair.

During the entire repair process, recruited stem cells produce paracrine factors. These paracrine factors exhibit antiapoptotic, proangiogenic, and accelerated cell proliferation effects, which are necessary to promote tissue regeneration.^{77, 78} Exogenous MSCs implanted in the injured areas can produce cytokines, such as fibroblast growth factor (FGF)-2, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-β through paracrine effects.⁷⁹ These cytokines promote angiogenesis as well as fibroblast migration and proliferation.⁸⁰ Moreover, they accelerate the deposition of collagen,⁸¹ regulate the inflammatory response in damaged skin tissues, and promote skin tissue regeneration.⁸²

Reportedly, treatment using paracrine factors (TGF-β, FGF-2, angiopoietin-2, and VEGF-1) secreted by transplanted bone marrow MSCs after myocardial infarction modeling in rats manipulated the microenvironment and reduced inflammation.⁸³ From this experiment, it can be concluded that the cytokines produced by stem cells at the infarct site can effectively promote angiogenesis and accelerate cell migration. In addition, a study conducted on mouse spinal cord MSCs by coculturing dermal fibroblasts with insert-grown bone marrow MSCs and cell scratching experiments showed that the paracrine effect of bone marrow-derived MSCs could accelerate the proliferation and migration of dermal fibroblasts, apart from the chemotaxis assay.⁸⁴ The migration of endogenous MSCs and endothelial progenitor cells has also been considered by researchers as an important mechanism for repairing damaged skin. Moreover, a study reported that MSCs of bone marrow and adipose origin could be isolated from a rat model of severe limb ischemia.⁸⁵ In vitro and in vivo experiments revealed that bone marrow-derived MSCs can promote endothelial cell migration, muscle reorganization, limb function improvement, and neovascularization. In addition, a previous study on rats has reported treatment of deep second-degree burns with bone marrow MSCs through paracrine effects via the stromal cell-derived factor (SDF)-1 alpha/CXCR4 pathway.⁸⁶ This approach induces the accumulation of endogenous MSCs and endothelial progenitor cells at the wound site, which can accelerate skin healing. Many of the therapeutic capabilities of stem cells rely on their paracrine actions. Various growth factors regulate the differentiation of fibroblasts and endothelial progenitor cells and promote their transfer to the wound. This mechanism promotes the formation of new blood vessels and granulation tissue, laying the foundation for skin regeneration.³⁶ Stem cell paracrine factors acting on damaged skin tissue are more effective than direct stem cell differentiation⁸⁷ and the healing is faster.⁸⁷ The potential clinical applications of cytokines derived from stem cells and the experimental models mentioned in the literature are listed in Table 2.

2.3 Stem cells promote bone tissue regeneration

Bone is a mineralized tissue that provides structural support to the vertebrate body.⁹¹ It protects internal organs, attaches to muscles to allow the body to move, and plays an important role in regulating serum calcium and phosphorus levels.⁹² The response of bone tissue to trauma is a continuous and complex process that includes an inflammatory response, activation of repair mechanisms, and remodeling of various tissues.⁹³ If a bone defect is followed by delayed healing or nonhealing that develops into a larger defect, traditional restorative means are no longer sufficient to restore such defects.^{94, 95} Regenerating damaged bone tissue to its predisease state has been a major challenge for clinicians and researchers worldwide.⁹⁶ Researchers aim to address this problem with new treatments.⁹⁷ Recently, stem cells have been found to be a good medium and option for achieving bone tissue regeneration.⁹⁸ The use of bone marrow MSCs in bone tissue engineering has entered the preclinical phase, and the U.S. Food and Drug Administration has approved an in vitro cell manufacturing procedure for obtaining biologically active bone marrow MSCs from high-quality human bone marrow.⁹² Stem cells can migrate to the site of injury and have the ability to differentiate into local components of the injury site and help regenerate the tissue.^99-102 For example, Kaku et al.⁸⁹ isolated MSCs from the bone marrow of rat femurs and transplanted them into a rat model with cranial defects. These results suggest that MSC transplantation may be a new option for cranioplasty. MSCs can not only repair bone tissue but also form suture-like gaps and promote cranial bone growth.

A direct reflection of the initial stage of bone injury is the formation of hematoma and the onset of an inflammatory response; hematoma can effectively prevent excess bleeding.¹⁰³ The fracture hematoma is initially infiltrated by immune cells, mainly neutrophils and macrophages.¹⁰⁴ When the bone tissue is damaged, blood vessels rupture, platelets are activated, and multiple coagulation factors, cytokines, and chemokines are released, triggering a hemostatic effect. Multiple cell types are recruited to migrate to the site of injury, contributing to the initial stage of bone tissue repair.¹⁰⁵ Inflammation is a protective response of tissues against harmful stimuli and leads to the initiation of the healing process.^{106, 107} Transplantation of stem cells into bone injury sites revealed the systemic anti-inflammatory effects of the cytokines they release.¹⁰⁸ The acute inflammatory phase peaks at 24−48 h after injury and subsides after one week. On the first day of a bone injury, neutrophils arrive at the injury site and release signaling molecules that recruit macrophages to the injury site.¹⁰⁹ Granero-Molto et al.¹⁰⁸ reported the presence of bone marrow MSCs in the region of new bone formation in a mouse fracture model. This indicated the promoting effect of stem cells on bone tissue regeneration. Macrophages not only engulf necrotic cells and tissue debris at the fracture site, but also initiate the recruitment of MSCs and vascular progenitor cells.^{110, 111} During acute inflammation, macrophages amplify the inflammatory response by phagocytosing invading microbes and recruiting additional immune cells to restore tissue homeostasis.¹¹² A study demonstrated that MSCs can mediate elevated M1 macrophage marker expression and promote M2 macrophage polarization, tissue vascularization, and bone volume increase in a rat femoral defect model.¹¹³ This study illustrated the anti-inflammatory and osteogenic effects of stem cells. Furthermore, it was found that BMSCs reversed the pure Lap-induced polarization of mouse-derived macrophages RAW 264.7 cells from M1 to M2 and promoted osteogenesis.¹¹⁴ These results suggested that BMSCs improved Lap-induced inflammation and enhanced bone formation. BMSCs were isolated from rats and cocultured with macrophages using hydrolyzed fish collagen, which inhibited the proliferation of PBMCs and significantly increased the expression levels of anti-inflammatory mediators, including IL-6, TGF-β1, and PGE2.¹¹⁵ BMSCs exhibit immunosuppressive capacity through crosstalk with immune cells, significantly limiting the inflammatory response after bone injury.¹¹⁶ Inflammatory cytokines play important role in regulating bone tissue destruction and promoting bone tissue regeneration.⁹⁰ Bone healing after an injury is a complex biological and biomechanical process,¹¹⁷ and final healing is highly dependent on the initial inflammatory phase, which is influenced by local and systemic responses to the injury stimulus. In addition, immune cells and mesenchymal stromal cells are involved in critical intercellular communication and crosstalk to regulate bone healing. Therefore, understanding the mechanisms of stem cells in bone healing is important for practical application.

It has been found that during the repair process, recruited stem cells secrete various chemokines, cytokines, and growth factors, which are collectively known as paracrine factors and are necessary to promote tissue repair and regeneration or tissue differentiation.^{65, 77, 87} Cytokines produced by stem cells through their paracrine action, including IL-1, IL-6, platelet-derived growth factor (PDGF), VEGF, and bone morphogenetic protein (BMP), play an essential role in bone tissue regeneration.¹¹⁸ These cytokines initiate the process of bone tissue formation and generate new blood vessels.

A study conducted by transplanting stem cells to an injured site revealed that the stem cells significantly reduced IL-6 levels on days 1 and 3 after the fracture, and reduced TNF-α and IL-1β levels on day 3.¹¹⁹ This process inhibits tissue damage and prevents the development of fibrosis, thereby promoting rapid bone tissue regeneration. In our previous study, we reported that the cytokines secreted by BMSCs treated with sinusoidal electromagnetic fields transplanted into a rat cranial defect model could better promote neoangiogenesis and bone immunomodulation, which play important roles in bone regeneration.¹²⁰ The osteogenic potential of BMSCs and the facilitative role of the paracrine function of BMSCs in promoting bone regeneration were illustrated. Pearson et al.¹²¹ implanted BMP-2 in a rat femoral defect model, which significantly and strongly induced neovascularization, and found BMP-2-induced angiogenesis through paracrine signals secreted by bone progenitor cells. The presence of cytokines such as insulin-like growth factor-1 (IGF-1), VEGF, TGF-β1, and HGF in MSC-CM significantly increased the migration and expression of genes associated with osteogenesis, and bone regeneration was observed in the rat cranial bone.¹²² In addition to traditional methods, some emerging cytokine techniques are gradually being developed. For example, Yu et al.¹²³ evaluated the bone regeneration effect of collagen membranes chemically coupled to SDF-1α in a rat model and found the chemical coupling to significantly promote the formation of new bone and microvasculature compared with the physical adsorption of SDF-1α. The study also provided a cell-free method to shorten bone healing time and improve the success rate of guided bone regeneration.

2.4 Other applications of stem cells in regenerative medicine

Apart from thoughts mentioned above, stem cells can promote regeneration of nerve and hair follicle tissues in addition to skin and bone tissues. Central nervous system diseases, such as stroke, traumatic brain injury, Alzheimer's disease (AD), and Parkinson's disease, are usually accompanied by neuroinflammation and nerve regeneration.¹²⁴ Nerve injury is one of the most common types of traumatic injury, commonly occurring in accidents and medically induced injuries.¹²⁵ However, traditional treatments are limited, which helps to find an alternative therapy is especially critical, and stem cell therapy is an excellent medium. Studies have shown that transplantation of stem cells are highly effective, both in terms of the number of axons and the restoration of function, showing the safety of this method.¹²⁶ In a short period of injury, the myelin sheath of the distal nerve dissociates, macrophages infiltrate and phagocytic debris produce an inflammatory response. Yang et al.¹²⁷ found that transplantation of undifferentiated tooth-derived stem cells (DSCs) could reduce the inflammatory response and promote neuronal regeneration by inhibiting the expression of IL-1 and member A of the Ras homologue gene family. Moreover, it has been found that transplanted NSCs reduce M1-type proinflammatory macrophage infiltration, promote M1-type to M2-type polarization, increase the release of anti-inflammatory factors (e.g., IL-10), and exert neuroprotective effects.¹²⁸ After stem cell transplantation, the stem cells are still able to produce and secrete various immune factors and neurotrophic factors to improve neurological function, even if they do not differentiate directly into nerve cells or mature nerve cells differentiated after limited transplantation.¹²⁹ Nerve regeneration requires the growth of nerve axons and secreted expression of neurotrophic factors, such as FGF-2, GDNF, and BDNF. A significant increase in the number of synapses was found after transplantation of stem cells to the injury site.¹³⁰ Studies have shown that stem cells are effective in treating neurological disorders, significantly promoting changes in neurological function.¹³¹ Vojnits et al.¹³² suggests transplantation of injury-induced MDSCs or their cell extracts into Duchenne muscular dystrophy mice (i.e., mdx mice) significantly promotes morphological and functional recovery of already injured neuromuscular junctions. In addition, umbilical cord MSCs (UC-MSCs) have multidirectional differentiation possibilities and improve cognitive dysfunction caused by AD after transplantation.¹³³ Stem cells for neural regeneration have a wide range of applications, but there are still many challenges for their clinical application, such as the lack of gold standard for cell isolation and culture, and preservation methods, genetic material variation and ethical issues. However, it is believed that these problems will be solved as the research progresses.

Stem cells are also a research hotspot in the field of hair follicle regeneration. Hair follicle is an organ with self-renewal ability and under normal conditions. The hair growth cycle goes through three phases—anagen phase, apoptosis phase, and resting phase to maintain the cycle for life.¹³⁴ However, due to the weak physical resistance of hair follicles and their susceptibility to genetic or environmental factors, leading to irreversible damage to hair follicles. Therefore, hair follicle regeneration has become an extremely important research area in the field of regenerative medicine. The role of stem cells in promoting hair follicle regeneration has become a hot issue.¹³⁵ Under normal physiological conditions, stem cells in each region maintain their own tissue renewal. When trauma occurs, stem cells located in the augmentation site can rapidly divide and proliferate to participate in the repair of the hair follicle.¹³⁶ ASCs are isolated from adipose tissue and induced to differentiate in dermal papilla formation medium for 30 days by visual and histological observation. The results of growth factor secretion were evaluated by immunohistochemistry and immunoblotting, showing that ASCs dermal papilla-like tissues (DPLTs) have characteristics of dermal papillae and have a positive effect on hair regeneration by secreting growth factors.¹³⁷ Stem cells can produce bioactive factors such as VEGF, IGF, and PDGF through paracrine action. When stem cells are applied to damaged hair follicles, these substances can regulate the hair growth cycle and promote hair growth.¹³⁸ Bu et al.¹³⁹ conducted a study in which cord blood MSCs were isolated and cultured in vitro and induced to differentiate into CK15 hair follicle cells. High speed induction of high purity differentiation into hair follicle cells was achieved by adding hair follicle extracellular matrix (ECM) to the culture medium. This study provides a new idea to promote the regeneration of hair follicle tissue and has a wide range of application prospects. And Fukuoka et al.¹⁴⁰ reported that ADSCs were isolated from the adipose tissue of healthy women, and the supernatant of ADSCs was collected and made into a lyophilized powder to act on the damaged hair follicles of the patients, and after 1−3 months of treatment, the number of hair follicles in the patients increased significantly. A previous study reported that mouse-derived pluripotent stem cells were effective in promoting hair follicle development.¹⁴¹ Stem cells have a broad application prospect in hair follicle regeneration since the development of regenerative medicine.

2.5 Clinical applications of stem cells

Regenerative medicine refers to the use of biological and engineering methods to promote repair and regeneration of damaged tissues, which restores the normal tissue characteristics and functions of an organism. The application of stem cells in regenerative medicine has gained widespread attention over the last 50 years. Because of their strong self-renewal and dividing abilities, stem cells are the most widely used cells in the field of tissue regeneration therapy and play an important role in tissue healing and regenerative medicine.¹⁴² According to our research, bone marrow MSCs, umbilical cord MSCs, and adipose MSCs have shown good ability to promote skin tissue regeneration.⁸⁸ Hassan, a 7-year-old Syrian “butterfly” boy, underwent treatment using his own skin cells to replace almost all his skin¹⁴³ and has since gained new life (Table 3). Stem cells have low immunogenicity and can be allografted or xenografted for clinical treatment. Exogenous stem cells are implanted to promote wound repair for skin regeneration.

Bone is a highly vascularized organ, and the skeletal system responds to external stimuli in numerous ways.¹⁴⁴ Repairing bone defects has always been an important clinical challenge, and innovations and improvements in bone repair are changing daily. Compared with traditional means of tissue repair, regenerative cellular and molecular techniques are improving, and the repair effect of stem cells on tissues is demonstrated by the ability to induce and stimulate differentiation into parenchymal cells in vitro to replace damaged areas.^{4, 145} There have been several clinical trials on stem cells for bone regeneration. Thus, the promotion of stem cell paracrine action on bone tissue regeneration is illustrated, and it is noteworthy that the paracrine function of stem cells has recently received more attention than the direct differentiation of stem cells.

3.1 Stem cell exosomes promote skin regeneration

Similar to stem cells, stem cell exosomes play different roles at different stages of wound healing. Exosomes can promote cell proliferation and neovascularization by regulating the inflammatory response¹⁶⁹ (Table 4). In addition, they can inhibit scarring and promote other mechanisms of effective skin tissue regeneration.^170-172

The inflammatory response is the initial stage of skin tissue regeneration can produce an immune response that includes proliferation of immune cells together with secretion of inflammatory and chemotactic factors.¹⁷⁷ A previous study reported that diabetic mice wounded by molding were treated by implantation of exogenous MSC exosomes. Exosomes were found to activate the PTEN/AKT signaling pathway to promote macrophage anti-inflammatory phenotype M2-type polarization and inhibited proinflammatory phenotype M1-type polarization. Furthermore, they inhibited the expression of proinflammatory factors IL-1β and TNF-α, promoting the expression of the anti-inflammatory factor IL-10, which accelerated the healing rate of skin injury in mice. This effectively demonstrated their ability to promote skin tissue regeneration.¹⁷³ Several investigators have reported that human exosomes derived from umbilical cord MSCs can mediate miR-181c expression and were found to effectively inhibit the TLR4 signaling pathway.¹⁷⁴ Exosomes can increase the expression of anti-inflammatory factors, which, in turn, reduce the inflammatory response to burn-induced skin damage in rats. Several researchers have conducted studies in which MSC-Exos were found to act as recruiters and trainers of immune cells, synergistically promoting the levels of beneficial macrophages and regulating T cells in the skin wounds of mice. They not only inhibited the proliferation of T lymphocytes but also promoted the conversion of activated T lymphocytes to regulatory T cells.¹⁷⁸ This inhibits the inflammatory reaction and promotes skin tissue regeneration. A moderate inflammatory response plays a role in fighting infection and removing damaged cells as well as broken cell debris; however, an excessive inflammatory response can prolong the healing cycle of skin wounds, with serious consequences.¹⁷⁹ Therefore, reducing the inflammatory response during wound healing is an important aspect of skin wound repair.¹⁸⁰

As the inflammatory response proceeds, there is a gradual transition from the inflammatory phase to the proliferative phase, in which leukocytes release cytokines, approximately 3−10 days after injury. This results in the induced migration of endothelial cells and fibroblasts to the wound center as well as the formation of new blood vessels and granulation tissue. The results of a study using MSCs in diabetic rats showed increased expression of CD31 and Ki67 in whole-skin wounds. This contributed to the enhanced regenerative capacity of granulation tissue and increased expression levels of VEGF and TGFβ-1.¹⁸¹ In addition, it made wound closure significantly faster and effectively promoted proliferation of skin tissue cells. Several studies have shown that miR-21 is highly expressed in the exosomes of adipose MSCs and can activate the PI3K/AKT signaling pathway to affect MMP-2 and TIMP-1 protein expression.^{175, 176} This significantly promotes accelerated wound healing. It enhances the proliferation of keratinocytes and fibroblasts while promoting the production of granulation tissue. Moreover, MSC-Exos were reported to significantly increase the level of angiopoietin 2 in wounds and enhance angiopoietin 2 proliferation, migration, and tube-forming abilities.¹⁸² This mechanism suggests that stem cell exosomes play an important role in the proliferative phase of skin tissue regeneration.

The last stage of skin regeneration is the remodeling phase, which is mainly characterized by a gradual decrease in the production of new blood vessels and gradual fibrosis of the granulation tissue. During this process, fibroblasts proliferate significantly, and the newly deposited collagen molecules cross-link, leading to an increase in the tensile strength of the tissue, which results in scar remodeling to restore the normal skin structure, a phase that can last for months to years.¹⁸³ In particular, the therapeutic effect of TSG-6-modified MSC-Exos was investigated in a mouse total wound model. The results demonstrated that exosomes from TSG-6-modified BMSCs inhibit scar formation by reducing inflammation and inhibiting collagen deposition.¹⁸⁴ According to a previous report, treatment of mice with umbilical cord-derived MSC-Exos was found to increase the density of myofibroblasts and inhibit myofibroblast accumulation after a period of time.¹⁸⁵ They have also been shown to reduce the somatic inflammatory response and scarring. Stem cell exosomes reduce scarring during the remodeling phase of skin tissue regeneration, mainly by inhibiting the accumulation of fibroblasts and suppressing collagen deposition.^186-188

In summary, stem cell exosomes are involved in the whole process of skin tissue regeneration and effectively regulate the inflammatory response. They cause proliferative differentiation of endothelial fibroblasts, which results in neoangiogenesis and the inhibition of scarring (Figures 1 and 2).

3.2 Stem cell exosomes promote bone regeneration

The regeneration of bone defects caused by trauma, infection, tumors, or inherent genetic diseases is a global clinical challenge. More than 10 million bone graft procedures are performed each year, and this number is growing at a rate of 10% annually.¹⁸⁹ Bone has a certain ability to regenerate; however, once the critical size is exceeded, spontaneous regeneration of bone is limited. Therefore, a substance is needed to promote bone regeneration. Bone repair and regeneration are complex processes, including tissue regeneration, angiogenesis, and immune regulation, which not only involve BMSCs, osteoblasts, osteoclasts, bone precursor cells, and other bone-related cells but also immune cells, vascular endothelial cells, and other system cells also play important roles.¹⁹⁰ Exosomes are tiny vesicles secreted by living cells^{191, 192} that carry relevant proteins, lipids, nucleic acids, and other substances derived from late endonucleosomal multivesicular bodies, which play an important role in intercellular information transfer.^193-195 Exosomes are products of the paracrine mechanism of stem cells, are more rapidly utilized than stem cells themselves, and have a wide range of clinical applications.^196-198 Regev-Rudzki N et al.¹⁹⁴ examined the therapeutic effects of MSC-Exos during fracture healing in a CD9−/− mouse model and found that MSC-Exos were effective in promoting fracture healing. Zhang et al.¹⁹⁹ reported results of implanting human embryonic-derived exosomes in a rat model of cartilage defect and found that exosome treatment resulted in the complete recovery of cartilage and subchondral bone by the 12th week. These two studies demonstrated the efficacy of stem cell exosomes in bone regeneration laying a foundation for subsequent studies, provided a guiding role, and showed potential of the stem cell exosomes for clinical application for bone regeneration.²⁰⁰ Thus, stem cell-derived exosomes have become a current research hotspot for tissue injury repair.

In the early stages of bone injury, the acute response to trauma produces a hematoma, preventing further serious trauma such as fractures.²⁰¹ An immune response ensues with the secretion of IL-1, IL-6, and TNF-α by neutrophils and macrophages during the first 24 h after the fracture, helping to initiate the repair process through cell recruitment.²⁰² An important immunomodulatory property of stem cell exosomes is the promotion of M2-type macrophage polarization and enhanced expression of anti-inflammatory cytokines, thereby reducing the local inflammatory response. This immune response is critical for the overall repair process. Lo Sicco et al.²⁰³ reported that adipose stem cells can secrete exosomes with anti-inflammatory effects when cocultured with macrophages and are effectively internalized, promoting macrophage proliferation and enhancing their polarization from M1 to M2 type in a short period of time. They also showed that MSC-Exos can effectively reduce the local inflammatory response in mice with skeletal muscle injury in in vivo experiments.²⁰³ This study confirmed the ability of MSC-Exos to regulate macrophage polarization, which may have potential applications in skeletal muscle tissue repair and regeneration. In an immunocompetent rat model, human embryonic MSC-Exos promoted chondrocyte proliferation and matrix synthesis, with more M2-type macrophage infiltration than M1-type macrophages at cartilage defects, along with the reduced expression of inflammatory cytokines IL-1β and TNF-α, thereby promoting cartilage repair.²⁰⁴ Zhang et al.²⁰⁵ examined the role of MSC-Exos in the regulation of the inflammatory response, nociceptive behavior, condylar cartilage and subchondral bone healing in an immunocompetent rat model of temporomandibular joint osteoarthritis. The observed exosome-mediated repair of osteoarthritic temporomandibular joint osteoarthritis was characterized by the early inhibition of pain and inflammation-reducing degeneration, followed by sustained proliferation.²⁰⁵ The results showed progressive improvement in matrix expression and subchondral bone structure in rats, leading to overall joint recovery and regeneration. Therefore, stem cell exosomes can promote tissue regeneration by modulating immune function, providing a basis for cell-free therapy. However, the mechanism of action of stem cell exosomes still requires thorough investigation before clinical trials can be conducted. Stem cell exosomes produce growth factors such as TGFβ, BMPs, IGF-1, PDGF, FGF2, and VEGF and chemokines such as monocyte chemotactic protein-1 and monocyte inflammatory protein-1a by paracrine action, all of which contribute to the stimulation of neoangiogenesis at the fracture site. Takeuchi et al.²⁰⁶ implanted human bone marrow-derived MSC-Exos in a rat cranial defect model. MSC-Exos enhanced cell migration and the expression of osteogenic and angiogenic genes in MSCs, compared with that in other groups by the fourth week after treatment. Histologically, the MSC-Exos group showed significant aggregation of osteoblast-like cells and vascular endothelial cells.²⁰⁶ This study illustrated that MSC-Exos promote angiogenesis and bone regeneration at an early stage. Considering the tissue regeneration of transplanted cells and their secretome it was concluded that exosomes might play an important role, especially in angiogenesis. Another study showed that transplantation of UMSC-Exos significantly enhanced angiogenesis and bone healing in a rat model of a femoral fracture.²⁰⁷ Implanted UMSC-Exos may be a key clinical strategy for accelerating fracture healing by promoting angiogenesis. Another study determined that huc-mscs-sev can promote osteogenesis and angiogenesis by delivering miR-23a-3p to activate the PTEN/AKT signaling pathway for vascularized bone regeneration.²⁰⁸ This study described the key issues associated with bone regeneration and reconstruction. In mouse and rat models, hard healing tissue remodeling begins 3−4 weeks after fracture and continues for months to years.²⁰⁹ As a cell-free agent, stem cell exosomes avoid both the immune rejection of stem cell therapy and ethical controversy and reflect a better prospect for application in tissue regeneration. As nanoparticles (NPs), exosomes can cross various barriers (e.g., blood-cerebrospinal fluid barrier, capillaries, etc.) and can be directly taken up by and act on target cells with higher action efficiency. Exosome therapy can effectively avoid safety issues associated with the direct application of stem cell therapy and is easy to prepare and transport. However, the mode, dose, and long-term safety of its application in humans have yet to be evaluated. As the mechanisms of action of stem cell exosomes are studied further, their effects and mechanisms on tissue regeneration will become clearer.

4.1 Polymeric nanoformulations

Polymeric NPs are efficient drug delivery carriers that can be divided into synthetic and natural polymers and have many outstanding pharmacokinetic properties. The polymeric nanoformulations bind to the stem cells and help the drug reach the designated site and exert its therapeutic effect. Researchers have identified NPs as potential candidates to regulate NSC differentiation, which currently allows stem cells to differentiate into various cell types.²²⁸ Autologous adipose-derived stem cells (ADSCs) can be safe and effective for the treatment of chronic myocardial ischemia and acute myocardial infarction, but neither ADSCs nor simvastatin-coupled nanoformulations alone can achieve the desired therapeutic effect.²²⁹ After systemic administration of both, a small number of porous polylactide-co-glycolide (PGLA)-NP-loaded ADSCs gradually release statin for tissue regeneration,²³⁰ significantly enhancing the therapeutic effect. To overcome the resistance to chemotherapy drugs, researchers have designed a NP containing RGD^{231, 232} and increased the efficacy of adriamycin against cancer stem cells (CSCs). NPs can enhance the transplantation potential of human hematopoietic stem cells,²³³ suggesting that NPs have the potential to overcome the current limitations of HSC gene editing. Chitosan medium could be used to mimic the microenvironment of CSCs, increase the expression of stem cell characterization-related genes²²⁰ and markers of CSCs to block differentiation and reduce the number of CSCs.

The combination of polymeric magnetic nanoformulations (MNPs) and exosomes provides new avenues for exosome-based nanotherapies. Local release of exosomes captured under acidic pH conditions by accumulation of nanoformulations as well as cleavage of hydrazine bonds in the presence of a local magnetic field.²³⁴ By encapsulating adriamycin (DOX) into isolated exosomes, MNPs are encapsulated to allow the accumulation of exosomes at the site of the lesion and subsequently the targeted release of exosomes.²³⁵ In combination, the drugs can kill cancer cells. Although MNPs can deliver anti-HIV drugs through an in vitro blood–brain barrier model, high doses of MNPs may damage cells. Correspondingly, exosomal EVs (xev) can cross the blood–brain barrier but lack long-range site specificity. When MNPs and xevs are combined as nanocarriers,²³⁶ they can target delivery of anti-HIV fusion agents effectively across the blood–brain barrier. Both MNPs and exosomes from stem cells have been shown to be effective in wound healing. The preparation of exosomes using BMSCs stimulated by MNPs in conjunction with static magnetic fields²³⁷ can further enhance wound repair.

4.2 Liposomes and nanoliposomes

Liposomes act as nanocarriers for targeted drug delivery for both hydrophilic and lipophilic drugs and improve drug solubility and stability.^{222, 223} Preparation of aspirin-containing liposomes by a thin film dispersion method and integration into DOPA3d printed PCL scaffold²³⁸ enhances PCL stents. It has been verified that CD146 + liposome magnetic beads could successfully separate CD146 and ADSCs,²³⁹ which can promote the repair of articular cartilage defects. If the liposome is injected directly into the bone cavity, TGF is released over several weeks. TGF-loaded liposome-coupled scaffolds exhibit enhanced release and localization. The subcutaneous implantation of hydroxyphosphate composite scaffolds coupled with bisphosphate-encapsulated liposomes in rats exhibited a strong binding effect, which increased drug retention.²⁴⁰ Long-circulating polyethylene glycolyzed poly (tritiated ester) liposomes induce CSCs apoptosis in mice and are effective therapeutic agents for further inhibition of CSCs growth.²⁴¹ Cross-linked multilayer liposomes composed of adriamycin (Dox) and salicin (Sal) interact with breast cancer tumor cells as well as CSCs and effectively inhibit the cancer cells.²⁴² Biocompatible nanoprobes consisting of liposome-indocyanine green hybrid vesicles for safe labeling of hMSCs can track cells after drug administration²⁴³ until the drug is fully effective.

The binding of liposomes to exosomes can improve their targeting and increase their retention time, apart from the other remarkable features. A hybrid therapeutic nanovesicle in the conjugate of exosomes, drug-loaded thermosensitive liposomes, and hGLV loaded with photothermal agents effectively targeted homologous tumors in mice resulting in excellent photothermal treatment and complete tumor elimination.²⁴⁴ The efficiency of exosome encapsulation of large nucleic acids was largely enhanced by mixing the exosomes with liposomes. Subsequently, the synthesized liposome exosome mixture efficiently encapsulated large plasmids, including CRISPR-Cas9 expression vectors.²⁴⁵ The instability in the transport of exosomes to tumor cells can be resolved by fusion delivery of exosomes and liposomes,²⁴⁶ which are effectively absorbed by tumor cells and then can be concentrated near the tumor to cause apoptosis.

4.3 Microspheres

Microspheres are the only effective means of introducing biological materials into real-time differentiated ESC culture. Microspheres as nanoagents can track MSCs, deliver small molecules, and promote stem cells.^{224, 225} The encapsulation of stem cells in microlets can be used as stem cell repair units.²⁴⁷ Microspheres in stirred suspension can preserve stem cells from human ADSCs (hASCs), which bind to microspheres for a period of time after higher levels of pluripotent markers expressed by hASCs,²⁴⁸ and improve the proliferation, colony formation, network formation, multimesenchymal differentiation, and regenerative capacity of stem cells. Injection of umbilical cord-derived MSCs microspheres with controlled particle size and cell encapsulation ability into the rat uterus²⁴⁹ indicated their nature in promoting the repair and regeneration of the endometrium. BMSCs in the presence of open porous poly (lactic-co-glycolic acid) microspheres can enhance cell proliferation in vitro and in vivo promoting cartilage regeneration.²⁵⁰ Sodium alginate-based Janus microspheres for targeted delivery of MSCs to cartilage were measured and found to be less toxic to MSCs²⁵¹ with good restorative ability.

Exosomes have isolation and detection limitations during cancer diagnosis; furthermore, progress in the studies on exosome analysis is slow. Researchers have discovered a microfluidic device-based method for exosome isolation and detection, realizing that the microsphere-mediated dielectrophoretic separation and immunoaffinity detection²⁵² improve the effectiveness of exosomes in tumor marker detection and cancer diagnosis. Exosomes have significant regenerative potential for bone tissue engineering; conversely, the therapeutic potential of exosomes is limited, and there is some attrition during delivery. The high biological activity of exosomes adsorbed onto the surface of porous PLGA injectable microspheres with bioinspired polydopamine (PDA) coating enables the delivery of exosomes to effectively induce tissue regeneration,²⁵³ thus facilitating the clinical translation of exosome-based therapies. Integration of a PLGA microsphere delivery platform into interpenetrating network hydrogels facilitates sustained delivery of MSC-Exos, thereby promoting endogenous AF repair,^{254, 255} illustrates the potential of exosomes for cell-free bioactive repair.

4.4 Hydrogels

The connection between hydrogels and stem cells is complex due to the involvement of various factors, such as porosity, different polymer types, stiffness, compatibility, and degradation. As a carrier of stem cells, the right type of hydrogel must be selected for encapsulation in different situations for the purpose of transporting stem cells.²²⁶ Hydrogel materials have good biocompatibility and biodegradability and can solve the problem of the low survival rate of stem cell transplantation after spinal cord injury (SCI).²⁵⁶ Cells encapsulated in hydrogel microcapsules containing a thin oil layer ensure high cell viability before the cells reach the area of inflammation, segmental rupture of the hydrogel with reduced stiffness in the process of movement, and delivery of UCB-MSCs to the sites with inflammation,²⁵⁷ thus releasing the active substances to eliminate the inflammation. The use of acrylic hyaluronic acid (HA) hydrogel microspheres, a biodegradable hydrogel, can deliver carriers in vivo and release cells to local targets.²⁵⁸ A study reported that to enhance the attachment ability and preserve bone marrow MSCs in hydrogels, the viability, density, and delivery of paracrine factors in hMSCs using agarose-carbohydrate hydrogels must be optimized.^{259, 260}

Hydrogels efficiently deliver exosomes to individual cells, maintaining the biological activity of exosomes while performing various repairs. Hydrogels can stimulate the release of MSC-Exos and heal better than exosomes or hydrogels alone,²⁶¹ suggesting that sustained release of exosomes and hydrogels can synergistically promote wound healing. The synthesis of exosome gels by immobilizing exosomes in peptide-modified adhesive hydrogels improved the retention and release of exosomes in injured spinal cord tissue.²⁶² Experiments have demonstrated that hydrogels significantly improve the residence time and stability of exosomes in vivo, and hydrogel-bound exosomes have enhanced endothelial protection and proangiogenic capacity in vitro.²⁶³ The combination of exosomes and hydrogels promotes wound healing, and hydrogels are effective in delivering exosomes and improving exosomal capacity.¹⁸¹

4.5 Polymeric micelles

Polymeric micelles are self-assembled from amphiphilic polymers in an easy-to-use manner to achieve optimal loading, stability, body circulation, and targeted delivery.²²⁷ Dexmedetomidine micelles can be internalized by osteoblast-like cells and rBMSCs, resulting in the release of dexmedetomidine.²⁶⁴ Ibuprofen esterase-responsive copolymer nanomicelles further enhance the adaptability of (NPPCs) in harsh environments and enhance their repair capacity.^{265, 266} Polymeric micelle drugs have higher cytotoxicity and inhibition of CSC colony formation than free drugs, and can inhibit tumor growth by CSCs.²⁶⁷ Polymeric micelles assayed for their effects on stem cell proliferation and differentiation²⁶⁸ showed that they promote adipogenesis, chondrogenesis, and osteogenesis of stem cells. A new micelle-assisted method for efficiently loading anticancer drugs into exosome-like vesicles has been discovered with improved tumor accumulation and retention.²⁶⁹ Using the tumor characteristics of MSCs, a drug transfer system based on MSCs was developed, and paclitaxel (PTX)-encapsulated HA poly (d, l-lactate-co-glycolide) polymer gel (PTX/HA-PLGA) micelles were used for treatment.²⁷⁰ The results showed that the overexpression of CD44 on the surface of MSCs and tumor cells not only increased the micellar load of PTX/HA-PLGA in MSCs but also promoted drug delivery between MSCs and adjacent cells.

In summary, both stem cells and exosomes have some defects, such as the transport and release of a single substance. Combinations with nanoformulations can compensate for the shortcomings in this area. Nanopreparations have different effects in different fields; for example, polymeric NPs enhance the stability of drugs, especially protein-based drugs, and have better slow and controlled release properties compared with liposomes. The interactions between nanoformulations, stem cells, and exosomes allow for more efficient transport and delivery of stem cells and facilitates exosome accumulation near a specified location, better utilization of stem cells and exosomes for tissue repair and other capabilities (Figure 3).

5.1 Scaffolds

Due to the hostile nature of the pathological microenvironment, specific conditions must be maintained to improve their function. During the transplantation process, the scaffold acts as a bridge, a guide for axonal growth, and a carrier for the transfer of stem cells, thus changing the microenvironment. Currently, neural and MSCs have been transplanted into biomaterial scaffolds, and experiments are underway to regenerate the spinal cord.^{271, 272} Natural and synthetic biomaterials provide a controlled microenvironment for cells, thereby accelerating their growth and differentiation, and enhancing their viability in the human body. Biological scaffold materials are primarily used to repair damaged or missing tissues and to reconstruct their functions. Structural reconstitution and good clinical outcomes have been reported using biological scaffolds by the mechanism of release or production of impact factors, incorporation of endogenous stem cells into the scaffold, and modulation of innate immune responses.²⁷³ The most common method of culturing stem cells is the two-dimensional plastic technique. This medium does not exhibit a good stem cell niche in humans; it is a very fine microenvironment that includes supporting stromal cells, the ECM, and growth factors. Therefore, researchers and clinicians are seeking the best stem cell formulation that can be used for research and clinical applications using three-dimensional technology. Three-dimensional culture technology can better simulate cell–cell and cell–matrix interactions.²⁷⁴ In addition to cells, different matrices and scaffolds can also be used to support complex tissues. The availability of modern technology can facilitate the expansion of cell and scaffold technology, thus providing a powerful tool for regenerative medicine.²⁷⁵In trauma or spinal fusion, scaffolding is the main bone repair method, combined with active molecules and stem cells. Cell-based bone repair techniques have been shown to be superior to conventional techniques. A previous study showed higher osteogenic efficiency of stem cells grown in a suitable three-dimensional scaffold support.⁴ Research has shown that three-dimensional biological scaffolds can support and mimic the in vivo environment and help stem cells differentiate into skeletal cells. Stem cells have a strong repair capacity during burn wound repair. Application of different types of stem cells can effectively improve wound healing. In human experiments, homing of stem cells to traumatized surfaces cause re-epithelialization, angiogenesis, granulation, inhibition of apoptosis, and regeneration of skin attachments, which reduces infection rates. Animal studies have shown that stem cells are effective in accelerating wound healing.²⁷⁶ The application of stem cells to burn wounds can significantly promote wound healing via scaffolding. The direct implantation of three-dimensional bioprinted stem cells into blood vessels has great potential, especially the production of organ and tissue substitutes.^{277, 278} Promoting the differentiation of stem cells using bioprinting technology allows scaffolds to be reshaped over a period of time.^{279, 280} The ability to tune bioprinting properties, which can be used to create stem cell carriers and take full advantage of cell pluripotency, is of great importance in biomaterials and bioengineering. Optimal transplantation or implantation can be achieved by combining stem cells with an artificial scaffold. The interaction between a stem cell and its scaffold has important implications for differentiation and health.²⁸¹ Stem cells from the cystic part of rat hair were used for scanning electron microscopy, using the method of extracting collagen preparation and scaffolding. The results indicated that the growth and changes in stem cells in both scaffolded and nonscaffolded states showed clear signs of cell differentiation, displaying larger cuboidal bodies with star-shaped nuclei. Scanning electron microscopy revealed large porosity, which facilitates cell attachment and growth.²⁸² Stem cells have a significant differentiation role in osteogenesis, whereas collagen scaffolds are a suitable matrix for cell growth and differentiation. There is potential for application of injectable Matrigel as a scaffold for BMP-transduced rat dental capsule stem cells/precursor cells (rDFSCs) to promote in vitro osteogenesis and in vivo ectopic bone formation.²⁸³ BMP9 significantly promoted osteogenic differentiation of rDFSCs, whereas Matrigel significantly promoted osteogenesis of rDFSCs induced by BMP. Biomaterial scaffolds combined with stem cell transplantation improve cell survival and differentiation efficiency.²⁸⁴ The use of natural and synthetic biomaterials can mimic the ECM, which promotes the differentiation of MDSCs and thus re-establishes the correct ecological niche of muscle stem cells, positively influencing muscle repair.²⁸⁵ The binding of scaffolds with cell growth factors and ECM molecules can promote cell adhesion, proliferation, and induction of osteogenesis, thus providing signals for cell transplantation and regeneration,²⁸⁶ offering tremendous opportunities for treating a wide range of diseases.

5.2 Microneedling

MN is a promising new technology that can be used to provide multiple drugs for the treatment of ischemic cardiomyopathy. By encapsulating cardiac stromal cells in fibrin gels and inoculating them on the MN matrix, polymeric MNs create “channels” between MNs and the host myocardium, allowing them to deliver regenerative factors to the damaged myocardium thereby accelerating cardiac repair.^{287, 288} The activation of hair follicle stem cells (HFSCs) can accelerate the regeneration of hair follicles, but difficulties remain in improving and managing them effectively. A keratin microneedle can be separated from the hair for the continuous delivery of HFSC-activated substances through the microneedle tip.^{289, 290} Studies have shown that microneedle devices, in combination with MSC-derived exosomes and small-molecule drugs, are effective in reducing doses and improving treatment efficiency. Exosomes derived from MSCs have shown promising results for the treatment of SCI. However, conventional two-dimensional culture methods result in stem cell loss from MSCs, which greatly limits the potential efficacy of in vitro secretions from MSCs. The three-dimensional exosome in vitro culture method is effective. Conventional exogenous drug therapy relies on repeated local injections, which cause secondary damage and are ineffective. Researchers have developed a three-dimensional exohydrogel hybrid microneedle array patch for the in situ repair of SCI.²⁹¹ It was shown that three-dimensional cultured bone marrow MSCs can maintain their stem cell properties, thus three-dimensional exosomes can effectively reduce SCI-induced inflammation and glial scarring. A novel core–shell HA MN patch with iron-MSC-derived artificial nanovesicles (Fe-MSC-NVs) and polydopamine NPs (PDA NPs) was encapsulated in a needle tip for wound healing. Fe-MSC-NVs contain multiple therapeutic cytokines encapsulated in the HA nucleus at the end of the MN to promote angiogenesis.²⁹² In vivo experiments have shown that Fe-MSC-NVs/PDA MN patches are effective in wound repair in patients with diabetes. Skin MN accelerates the process of skin regeneration by creating a large amount of microdamage, which occurs when the skin is punctured by a very fine needle. To accelerate postoperative regeneration or to improve efficacy, MN is combined with drugs, vitamins, or stem cells.²⁹³ Owing to its high efficacy, few side effects, and short recovery period, MN has been widely used in clinical practice. MSCs are injected by encapsulating them in biomaterials, thus enhancing their stability. However, because of its poor efficacy, numerous cells are required for treatment. In addition, injection to the target site achieved using conventional syringes by local injection can cause significant damage. Researchers have developed a separable hybrid microneedle library (d-HMND) cell delivery system²⁹⁴ that can effectively inject MSCs locally. Researchers have invented a detachable microneedle patch, whose main component is chitosan lactate (CL) with adipose stem cell exosomes (EXO). CL and EXO synergistically promote hair regrowth and regulate the circulation of hair follicles.²⁹⁵ Animal studies have shown that drug-free microneedle patches significantly promote hair regrowth within 7 days, compared with the typically administered minoxidil at lower doses.^{296, 297} Microneedles deliver stem cell secretomes directly to the site of injury, demonstrating the relative therapeutic benefits of live cells while avoiding potential limitations.²⁹⁸ To improve the efficacy, a variety of biomaterials have been developed for the continuous and controlled delivery of stem cell secretomes. Direct delivery of stem cells and therapeutic macromolecules to the cardiac tissue and vascular smooth muscle cells²⁹⁹ breaks through the nonpermeable barrier and allows the drug to go directly to the target area for maximum effect, thereby reducing the required dose. In recent years, MSCs and MN techniques have been widely used to delay aging. To investigate the effect of human umbilical cord tissue matrix (hUC-MSC-CM) on skin brightness and rejuvenation, researchers conducted a clinical trial in which HUC-MSC-CM in combination with MN was found to delay aging and could be applied to facial rejuvenation by evaluating the volunteers’ skin radiance and texture as well as their self-satisfaction.³⁰⁰

5.3 Hydrogel scaffolds

Recently, applied ADSCs have been investigated, and biologically active injectable hydrogels have been developed as cell-transfer carriers. Hydrogel scaffolds are promising regenerative materials that can mimic the ECM and deliver signaling molecules.^{301, 302} The bionic injectable hydrogel mimics the natural microenvironment and provides the correct biochemical information for tissue regeneration.³⁰³ Researchers have developed amniotic tissue hydrogels as a delivery system for adipose MSCs and retained the stem cells at the target site, which could serve as a potential stem cell carrier that could be used and developed as a potential therapeutic agent for osteoarthritis.^304-306 ADSCs have better anti-inflammatory, chondroprotective, and paracrine functions that can be enhanced by genetic alterations. Direct cell transport without matrix support usually results in poor survival of the treated cells. Researchers have used bone grafting via genetically engineered ADSCs.³⁰⁷ An injectable ECM-mimetic hydrogel was developed as a cell-transfer vehicle to create a favorable microenvironment for ADSC spreading and proliferation.³⁰⁸ Hydrogels that mimic the ECM can reduce cell death during and after injection. Currently, self-healing natural hydrogels suffer from poor stiffness, low healing efficiency, poor biocompatibility, and poor hydrolytic stability in the treatment of SCIs. Researchers have developed an injectable self-healing HA gel based on multiple dynamic covalent bonds.³⁰⁹ This injectable, self-healing HA hydrogel is a promising material for nerve repair. Targeted transport of stem cells using scaffolds, microneedles, and injectable hydrogels can effectively repair lesion sites and maintain stem cells.