Regenerative Medicine in Gynecology

Abstract

The female reproductive tract undergoes dynamic changes across the lifespan. Congenital abnormalities, life events, and medical interventions can negatively impact the structure and function of reproductive tract organs resulting in life-long sequelae. The objective of regenerative gynecology is to discover and promote endogenous mechanisms by which a healthy tissue maintains overall tissue integrity after injury, disease, or with age. In this review, we discuss some of the key state of the art cell-based and scaffolding therapies that have been applied to regenerate gynecologic tissues and organs primarily in animal and tissue culture models. We further discuss the limitations of current technologies, problems of implementation and scalability, and the future outlook of the field.

Précis

In this review, we discuss current cell-based and scaffolding therapies applied to regenerative medicine in gynecology and the future outlook of the field.

Introduction

The female reproductive tract is a highly dynamic hormonally responsive system that significantly changes across the lifespan. Congenital absence of an organ, surgical removal, or tissue damaging treatments such as chemotherapy and radiation can lead to loss of structural and physiologic integrity of the reproductive organs and tissues. In addition, normal life events such as pregnancy, vaginal birth, menopause, and aging can result in substantial, life altering changes to the reproductive tract.

Virtually, all tissues in in the body have ability to remodel and heal. However, when remodeling capacity is incomplete, scarring ensues which leads to loss of function and other detrimental systemic effects across the lifespan. Lower order vertebrates including planarians, urodeles, fish and reptiles, fully regenerate tissue, entire organs, or limbs, leading to a complete resolution of injuries and restoration of normal structure and function following injury. Mammals, however, have a limited regenerative capacity which favors rapid resolution of healing rather than structural and functional restoration¹. This loss of a complete regenerative response in higher order species is an evolutionary tradeoff of an increasingly complex immune system that favors the formation of non-functional fibrotic tissue that comprises a scar².

Scarring following reproductive tract injury displays negative life-long sequelae. For example, vaginal birth, cesarean delivery, and reproductive tract surgeries may result in scarring that increases risk of pelvic organ prolapse, intrauterine adhesions, abnormal placentation, and infertility^3–5. Scarless wound healing has been observed in fetal mammals; however, regeneration is age dependent. In human development, tissue type, wound size, and gestational age are all factors that influence the transition from scarless regeneration to scar formation⁶. The phenotype of adult wound healing displays an intrinsic difference in host response. For example, the tissue scaffolding in adult tissue repair favors type 1 collagen which is non-elastic and provides mechanical strength and rigidity but impedes cellular migration. Whereas fetal wound healing displays a predominance of type III collagen deposition which is conducive for cell migration and proliferation. Extracellular matrix (ECM) components including hyaluronic acid and matrix metalloproteinases are reduced in adult wound healing promoting dehydration and accumulation of collagen. Adult wound healing is also considered pro-inflammatory with infiltration of many immune cell types and a rapid increase in pro-inflammatory cytokine expression including IL-6 and IL-8 whereas fetal healing is anti-inflammatory with increased IL-10 expression. Finally fetal tissue regeneration is mediated by rapid up-regulation of genes involved in cell growth and proliferation in addition to plentiful progenitor cells present and migrating to the site of injury⁶. Although the exact mechanisms of fetal tissue regeneration are still unknown, the constructive, scarless nature is due to differences in the ECM, inflammatory response, population of stem and niche cells present.

The goal of regenerative medicine is to redirect the default host response from destructive, scar formation to constructive, rejuvenation and repair. The objective of our field is to discover and promote endogenous mechanisms of a healthy tissue to maintain overall tissue integrity after injury, disease, or with age. As such, regenerative strategies are being investigated to treat or replace compromised organs and tissues. Here we will focus on cell-based and scaffolding therapies, their application in regenerative gynecology, limitations, and the future outlook of the field.

Cell-based Therapies

Platelet-rich Plasma (PRP)

The wound healing process is mediated by growth factors released by alpha granules from platelets. Platelet-rich plasma (PRP) is rich in chemo- and mitogenic factors that promote angiogenesis, cell proliferation, and wound healing⁷. PRP is a beneficial cell therapy because it is autologous with no risk of host immune response or transmission of donor micro-organisms. PRP has been used in gynecologic regenerative medicine including treatment of thinned endometrium, Asherman’s syndrome, premature ovarian failure, stress urinary incontinence, lichen sclerosis, and genitourinary fistula^8,9. Small cases series have demonstrated PRP intrauterine infusion increases endometrial thickness by 20% compared to controls^10,11. Further, 73.7% of women (n=19), aged 33–45, undergoing fertility treatment who experience refractory endometrium or thinning after hormone stimulation, had a positive pregnancy test after 2 interventions of PRP prior to embryo transfer^10,. While PRP treatment is promising in promoting tissue biostimulation, there are many limitations to adopting PRP use including a lack of standardization of preparation techniques and an absence of definitive benefits derived from prospective randomized clinical trials. Additional studies are needed to demonstrate if patient specific factors impact PRP quality, composition, and clinical outcome.

Stem cells

Stem cells are defined by their ability to self-renew, proliferate, and give rise to differentiated cell types¹². Stem cells have different degrees of regenerative capacity referred to as potency based on the cell’s degree of differentiation. For example, a zygote or fertilized egg is a totipotent stem cell as it can give rise to both the embryo and extraembryonic structures like the placenta. The cells of the embryo divide into the blastocyst as defined by the inner cell mass which contains embryonic stem cells which are pluripotent and give rise to all fetal tissues¹³. Embryonic stem cells (ESCs) give rise to multipotent stem cells such as mesenchymal stem cells or hematopoietic stem cells which have multiple downstream lineages. Adult stem cells are retained throughout life to maintain tissue health and orchestrate repair in the context of an injury¹⁴.

The acceptance of human ESCs faces hurdles, including ethical concerns regarding the use of human embryos and immune rejection after transplantation. Inducible pluripotent stem cells (iPSCs) have been developed to overcome these limitations. iPSCs are derived from adult somatic cells like skin fibroblasts and can be reprogrammed into a pluripotent-like state with similar potency to embryonic stem cells by activating transcription factors including oct3/4, sox2, klf4, and c-myc¹⁵. With a targeted cocktail of growth factors, iPSCS can be differentiated into the desired target cell type and injected back into the host at an injured site. For example, iPSC generated smooth muscle progenitor cells have been used to treat urethral sphincter dysfunction in a rat model of stress Specifically, smooth muscle cells derived from human iPSCs increase native tissue matrix remodeling by promoting elastin and collagen III content similar to transplanted smooth muscle cells derived from human ESCs¹⁶.. The benefit of using iPSCs in gynecologic regenerative medicine is the autologous nature. However, iPSC generation is slow. The use of donor cells from biobanks is possible but human leukocyte antigen (HLA) variability is a potential pitfall. Further, iPSC-derived cells or tissue could retain unchecked proliferation activity or immature cells after transplantation that can lead to tumor formation, most often teratomas^17,18. Purification of iPSC derivatives is one approach to reduce the heterogenicity and potential tumorigenicity. Magnetic-activated cell sorting (MACS) using a differentiated smooth muscle cell surface marker, CD34, significantly reduced the percentage of undifferentiated cells derived from iPSC and illustrates a path forward for reduced tumorigenicity in iPSC therapeutics¹⁹.

Mesenchymal stem cells (MSCs) are present in most adult tissues including bone marrow, adipose, endometrium, umbilical cord, amniotic fluid, placenta and menstrual blood²⁰. MSCs have improved in vitro culture and expansion capacity and reduced ethical concerns compared to iPSCs^21,22. MSCs have been used as a therapy in a wide variety of gynecologic conditions including Asherman syndrome, thinned endometrium, vulvovaginal atrophy, lichen sclerosis among others²³. MSCs exhibit anti-inflammatory effects, are migratory, and secrete growth factors²⁴. MSC transplant in a rat model of pelvic floor dysfunction show MSC migration to the injury site and were present in the healed vagina for 30 days after systemic transplantation²⁵. In a primate model of ovariectomy induced vaginal deterioration, MSC transplantation promoted ECM remodeling by increasing elastin and collagen I in the lamina propria of the vagina, increased smooth muscle in the muscularis, and improved biomechanical properties²⁶.

Adult or somatic stem cells are retained from organogenesis throughout the lifespan for tissue maintenance and repair. These cells are generally activated in the context of tissue injury and can both self-renew and generate the differentiated cell types that are needed to orchestrate tissue repair. Resident stem cells are present in multiple adult tissues including stomach, intestine, and liver and provide unique insight into organ-specific tissue repair^27–30. In the context of human reproduction, the endometrium undergoes dramatic tissue regeneration during the menstrual cycle. Endometrial regrowth has been observed in patients who underwent electrosurgical ablation of the endometrium for treatment of abnormal uterine bleeding suggesting resident cells were present that retain regenerative capacity³¹. As such, both stromal-like and epithelial stem cells have been proposed to repopulate the functional endometrial layer in response to endocrine stimulation³². Further these cells have been isolated and cultured to demonstrate self-renewal and multilineage differentiation. Endometrial stem cells have been used in mouse models of endometrial injury and have demonstrated accelerated tissue repair, cell proliferation, angiogenesis and increased pregnancy rate^33,34.

Harnessing stem cell function is the basis for cell and tissue regeneration and is a powerful tool in regenerative medicine for both tissue and organ replacement. However, stem cells are difficult to grow and maintain in culture and require a large number of cells to feasibly transplant. Moreover, the main benefits of stem cell transplantation have been derived from their anti-inflammatory properties with little evidence for engraftment and proliferation at the site of injury. This failure of translation into a regenerative benefit is likely due to a failure to recapitulate the stem cell niche – a highly specialized microenvironment in which a stem cell resides. The niche regulates stem cell function and fate signaling it to remain quiescent, to proliferate, or to differentiate. Further while the stemness is the key to tissue repair, stem cell source is controversial and stem cell transplant poses a risk for aberrant cell proliferation.

Synthetic scaffolds

Tissue interactive synthetic meshes

Synthetic scaffolding materials or surgical meshes are designed to act in place of native tissues to provide the mechanical and load-bearing support, such as that needed for repair of pelvic organ prolapse (POP). Unfortunately, current technologies are based on the premise of inertness with polymers intended to be nonreactive, noncarcinogenic, and nontoxic to host soft tissues. However, use of synthetic materials in gynecologic medicine has illustrated that all inert materials are recognized by the host immune system as foreign resulting in a robust foreign response with a predominance of M1 proinflammatory macrophages and myofibroblasts³⁵. Myofibroblasts deposit collagen and make contractile fibers allowing them to pull on tissue leading to fibrosis and pain. One example of this response is seen when silicone breast implants are encapsulated by myofibroblasts which can lead to capsule contraction to an extent that the implant is distorted and painful requiring removal³⁶. Urogynecologic polypropylene meshes behave similarly³⁷. A biomechanical mismatch occurs between mesh devices and the vaginal tissue which leads to tissue degeneration and atrophy as the vagina is “shielded’ from the stresses it needs to maintain its structure and function. Micromotion induces repetitive cycles of injury and repair triggering chronic inflammation and pain. Unstable mesh geometries under tension cause the pores of the mesh to collapse and the whole device to wrinkle resulting in uneven stress distributions across the vagina. Recent data suggests that in high stress areas, the myofibroblast predominates resulting in scarring, fibrosis, and pain while in low stress areas (beneath a wrinkle) the macrophage predominates initiating in tissue degradation and thinning, predisposing the tissue for mesh exposure³⁸.

The future of synthetic scaffolding in regenerative medicine will move away from inert materials that compromise already injured tissues and promote a constructive host response. One emerging engineering approach is the use of softer elastomeric meshes with geometries that are stable with loading³⁹. Auxetic materials have a negative Poisson’s ratio, which describes the ability of a material to expand with increased load bearing rather than collapse. The benefit of auxetic materials in POP repair is mesh pores will remain open when loaded which afford native tissue ingrowth⁴⁰.

Bioscaffolds

Acellular extracellular matrix (ECM) bioscaffolds most often derived from pigs have been a powerful cell free technology in regenerative medicine. The ECM is the product of cell secretions that organize into a 3-dimensional structure that supports tissue anchoring, cell adhesion, communication, and migration. The benefit of utilizing ECM as a template for tissue regeneration is that it can recreate the cellular environment of native tissue. ECM is biointeractive and will degrade after implantation to release bioactive peptides and growth factors that promote a constructive remodeling response. ECM bioscaffolds derived from urinary bladder, have demonstrated improved tissue regeneration in POP model in rhesus macaques⁴¹. Newly formed connective tissue attachments between the vagina and pelvic side wall were present following bladder ECM bioscaffold transplantation. Further when compared to traditional polypropylene mesh implants, bladder ECM implanted vagina displayed preservation of smooth muscle and collagen content and decreased vaginal stiffness. In addition, the population of anti-inflammatory M2 macrophages was increased in ECM transplants compared to polypropylene mesh suggesting a promotion of constructive host response⁴¹. Although additional studies are needed, ECM implantation as a bioscaffold for tissue regeneration is a promising technology for tissue repair in a POP model.

The native ECM provides the structural support for 3-dimensional tissue and its composition and mechanical stiffness direct cell proliferation and differentiation. Collagen is primarily secreted by fibroblasts. In adult wound healing, myofibroblasts are pro-inflammatory and are important for secreting collagen quickly after injury to provide a provisional matrix and support for immune cell migration⁴². However, in chronically injured or aged tissue, myofibroblasts can persist in tissues long after the healing response should have resolved creating a chronic inflammatory environment with excess ECM matrix deposition leading to fibrotic stiff tissues^43,44. Altering the ECM composition during wound healing has potential to improve tissue regeneration in adult tissue by redirecting the default host response to an anti-inflammatory constructive repair mechanism. ECM can be isolated from tissue through decellularization, which removes DNA and RNA but leaves behind the ECM components. ECM can be processed and rehydrated to create a hydrogel, a 3D bioscaffold support that can be used as a gel for bio-ink printing⁴⁵. Vaginal birth injury is associated with pelvic muscle atrophy, fibrosis, increased ECM deposition and a persistent inflammatory response leading to POP. In a rat model of birth injury, injection of an acellular ECM hydrogel derived from decellularized porcine skeletal muscle, at the time of injury or 4 weeks after injury improved pelvic floor muscle regeneration. Hydrogel injection was associated with myogenesis, reduced atrophy, and decreased expression of profibrotic genes⁴⁶. Hydrogels can provide conducive microenvironment for tissue repair that harnessing the endogenous properties ECM.

Discussion

Significant advances in both cell- and scaffold-based therapies have been made in regenerative gynecologic medicine which have expanded the field’s understanding of the constructive and destructive pathways present during tissue remodeling in adults. However, many of the approaches discussed have limitations in ethical use and scalability in human patients. Nevertheless, understanding the fundamental mechanisms of tissue repair in the pelvic organs and their associated tissues is necessary to inform future therapeutics.

Current investigations focus primarily on either the cells or the scaffold; however, it is well established that the microenvironment informs cell identity and function and vice versa. Biomimicry of the healthy microenvironment is an emerging approach in which bioscaffolds and repair devices are functionalized by incorporating peptide factors that direct cell signaling and stimulate a constructive remodeling response⁴⁷. Functionalization by conjugating synthetic and decellularized hydrogel bioscaffolds with growth and myogenic factors have shown promising regenerative capacity in the context of bone, cartilage, muscle, and bladder tissue repair^48–51. For example, when insulin-like growth factor-1 (IGF-1) is covalently bound to a fibrin-collagen scaffold and transplanted into a rat model of bladder repair, the bladder showed re-urothelialization after surgery with an IGF-1 dose-dependent increase in host smooth muscle cell formation⁵².

Newer technologies explore harnessing both the “seeds” and the “soil” to improve regenerative medicine outcomes by combining bioscaffold and stem cell technologies. For example, normal pregnancy and fetal development was observed in a rat transplanted with a decellularized uterine scaffold seeded with primary uterine cells or bone marrow-derived MSCs⁵³. However, pregnancy outcomes were influenced by the decellularization procedure which suggest that critical components of the ECM are needed for effective reconstruction. Alternative scaffolds including a porcine intestinal submucosa demonstrated effective niche environment for umbilical cord mesenchymal stem cell proliferation and adhesion and depending on the resection length, uterine repair and pregnancy outcomes were similar to control animals⁵⁴. In humans, a pilot study of young women with vaginal agenesis in whom a neovagina was created surgically using vulvar derived autologous epithelial and muscle cells seeded onto a biodegradable scaffolds had evidence of both vaginal epithelial and muscle layers in post operative biopsies⁵⁵. While these studies are promising, larger, well-controlled studies and a move toward biomimetic bioinspired solutions are needed to address the cell and environmental (scaffold) components for optimal tissue regeneration and repair.

Finally, it is still not known which reproductive organs house adult stem cells. The optimal approach for tissue bioengineering would include reaction of reconstructed tissue from native ECM and with endogenous stem cells. While adult stem cells have been confirmed in the placenta and endometrium, investigating if other reproductive organs, like the vagina and fallopian tube, contain stem cells is imperative^56–60. This information will expand the knowledge gap of basic tissue repair in these organs but also could provide a rich source of endogenous cells types to improve cell-based therapy and tissue regeneration.