Sports Medicine Applications

Journal Pre-proof Stem cells for treatment of musculoskeletal conditions orthopedic/ sports medicine applications

Mimi Zumwalt, Arubala P. Reddy PII:

S0925-4439(19)30352-7

DOI:

https://doi.org/10.1016/j.bbadis.2019.165624

Reference:

BBADIS 165624

To appear in:

BBA - Molecular Basis of Disease

Received date:

31 January 2019

Revised date:

22 November 2019

Accepted date:

23 November 2019

Please cite this article as: M. Zumwalt and A.P. Reddy, Stem cells for treatment of musculoskeletal conditions orthopedic/sports medicine applications, BBA - Molecular Basis of Disease(2019), https://doi.org/10.1016/j.bbadis.2019.165624

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© 2019 Published by Elsevier.

Journal Pre-proof Stem Cells for Treatment of Musculoskeletal Conditions Orthopedic/Sports Medicine Applications Mimi Zumwalt 1, Arubala P.Reddy 2 1.Tenured Professor of Orthopedic Surgery, Director of Sports Medicine, Texas Tech University health and Sciences center, 3601 4th street Lubbock, TX 2. Research assistant Professor, Nutritional Sciences /Human Sciencess Department, Texas Tech University 1301 Akron Ave Lubbock Texas 79409

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Abstract A myriad of musculoskeletal conditions afflicts a vast number of the world’s population from birth to death. Countless pathological diseases and traumatic injuries (acute and chronic) contribute to different human disabilities, causing a tremendous financial toll on the economy of healthcare. The medical field is continually searching for novel ways to combat orthopedically related conditions. The immediate goal is the restoration of anatomy then ultimately return of function in hopes of enhancing quality if not the quantity of life. Traditional methods involve surgical correction/reconstruction of skeletal deformities from fractures/soft tissue damage/ruptures or replacement/resection of degenerated joints. Modern research is currently concentrating on innovative procedures to replenish/restore the human body close to its original/natural state (1,2).

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Keywords: regenerative medicine, tissue engineering, stem cells, musculoskeletal disorders, orthopedic sports medicine, sex/gender, biomarkers, clinical trials.

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Introduction Musculoskeletal disorders (MSDs) due to injuries of joints, muscles, ligaments, nerves, and tendons affect the quality of life. According to the WHO report is the leading cause of worldwide disability. MSDs caused by older age; one in three and one in five adults suffer from painful, debilitating conditions. MSDs define by 150 variant diagnosis significantly reduce everyday life, increase pain, and discomforts in noncancerous conditions (3). The study conducted in 2017 provides evidence of the Global burden of the disease (GBD) finds MSDs contribute to global disability, and 16% of the adult in the peak earning years of their life suffer from disabilities. In America, every other adult (126.6 million) live with the musculoskeletal condition as chronic diseases like cardiovascular and respiratory diseases combined. The MSDs conditions treatment and cost rose up to $874 billion from 2011 cost $213 billion (4,5). Current demand in MSDs treatment is more research, preventative options, nutritional; mental wellbeing help is the few options to improve quality of life. Multiple pathologies linked to MSDs cannot be resolved with conservative management or surgical intervention. Tissue engineering, a branch of biomedical engineering and regenerative medicine, aims to replace/regenerate cells, tissues, and organs to rebuild bodily structures to regain their nascent biological condition. Stem cells, in particular, possess unique properties enabling them to differentiate into different types of tissue to treat a variety of musculoskeletal entities. To maximize positive results from stem cell therapy in modern medicine, researchers must also delve into identifying inherent sex differences in their inherent characteristics for the enhancement of cell-based therapies and clinical

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Journal Pre-proof application. Besides, the size/age/gender of both donors and recipient hosts also can affect treatment outcomes thus must be considered as well. This review will focus on stem cells and their potential in addressing more common orthopedic joint disorders related to aging, such as osteoarthritis/osteoporosis, along with treating traumatic events involving bones, muscles, tendons, and ligaments (1,2,3,6,7).

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History/Timeline Stem cell research originated back in the mid to late 1800s when scientists attempted fertilization of mammalian eggs ex-vivo along with discovering the regenerative capacity of specific cells (1,2,3,6,7). Researchers then discovered in the early 1900s the very first real “stem cells” (coined by a Russian histologist) upon finding out that blood cells could generate from other cells. Six decades later, successful transplantation of bone marrow occurred from one sibling to another (7). In the same decade (the 1960s), in vitro fertilization (IVF) of the first human egg occurred (6). Then in the next decade (the 1970s), England and Australia introduced the first baby born by IVF, along with human cord blood being found to yield hematopoietic stem cells (6,7). Subsequently, two decades later, in the late 1990s, the first human embryonic stem (ES) cell line was derived in the US. A decade after that (in the mid-late 2000s), British researchers claimed to have created embryonic-like stem cells (different categories) from umbilical cord blood. Compared to adult stem cells, cells from umbilical cord blood appeared to be able to differentiate into more cell types, possibly widening the field of cell-based therapeutics. Besides, another source of stem cells was isolated from amniotic fluid, lending a better alternative since the usage of ES cells was deemed controversial. At about the same time (2005), Korean scientists reportedly used unfertilized human oocytes to produce several human ES cell lines (7). Similarly, in 2000, before the discovery in Korea, researchers in Australia and Singapore were using blastocyst inner cell mass (donation from couples receiving infertility treatment) to create human ES cells (6). Then about ten years ago, the first successful human knee cartilage regeneration was reported using autologous adult mesenchymal stem cells (MSCs). Since that time, the creation of patient specific “induced pluripotent stem cells (iPSC) and culturing of human embryonic stem cells (ESCs) have been possible as well (8). Paralleling the steady rise of scientific research on stem cells, religious groups and government officials weighed in on potentially controversial debates in the public eye. Subsequently, starting in the mid-70’s ethical and governing boards created. Recently, these academies and the like continue to add amendments to originally proposed guidelines to maximize national policies with laws/procedures to protect the public while encouraging scientific advancement (9). Define guidelines helped to pave the way that federal funding will use ethically for harvesting and growing stem cells to develop medical treatment through clinical research (8). Definition Stem cells are present in almost all multi-cellular organisms. A stem cell is generic and possesses potency, capable of making an identical copy of itself over and over indefinitely, i.e., duplication/mitotic cell division. Stem cells can then go through differentiation/specialization into other cell types and ultimately creating numerous

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uniquely different bodily tissues (8). Specifically, they are progenitor cells that replace dead cells as part of regular turnover in the body. Three characteristics define stem cells: staying quiescent/undifferentiated until activated, capable of differentiation into multiple tissue types, and continual cell replications/self-renewal (10). They hold an extremely vital role in the growth, development, maintenance, and repair of all organs in the human body. By studying in-depth the essential properties of stem cells, scientists can perform research in the laboratory about potential new drugs and possible causes of congenital disabilities among other disease entities to help enhance the medical management of patients via modern healthcare (8). However, the definition of stem cells is better characterized as an essential portion of the more full tissue engineering field with four delineations: cellular/precursor production; conduction/delivery by scaffolds; induction/stimulation via cell surface markers/receptor molecules/growth factors; and mechanical activation through pathologic/physiologic loading (10).

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Classification Potency and sources are two ways by which stem cells can be classified. The above addresses a stem cell is differentiating capabilities into various types of cells: totipotent-all; pluripotent-most; toti- or multipotent - closely related cell family; oligopotent - few; and unipotent - cellular production of self/same type. The latter’s classifying system describes donor site(s) from where they originate (before or after birth), i.e., three general categories: embryos, adult, and induced pluripotent stem cells (iPSCs) (1,8,10).

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Embryonic stem cells (ESCs) Human ESCs were fist derived in 1998 from preimplantation embryos by prolonged undifferentiated proliferation and potential to develop three embryonic germ layer distinct development (11). Figure 1:

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Adult stem cells (ASCs) As compared to ESCs, which only found in one place, multi- or totipotent adult stem cells (ASCs) can be located post-natal in several areas within the human body. The function of these cells is for maintenance and reparation of their damaged, mature tissue. With a few exceptions, these ASCs are tissue-specific, i.e., they divide then multiply in response to cell death, regenerating the same type of tissues (1,8,10). The other nonspecific adult stem cells are derivatives of adipose tissue and umbilical cord (Wharton’s Jelly) (1,14). Finally, stem cells harvested from amniotic fluid or placenta have characteristics somewhere in between ESCs and ASCs since they are capable of self-renewal AND can differentiate into different types of cells (1). Figure 2:

Induced pluripotent stem cells (iPSC)

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Journal Pre-proof Stem cell research received a dramatic discovery in Japan a decade ago (2006) regarding the manipulation of cellular identity (1,15). This iPSC is reprogrammed by scientists using specialized adult somatic cells, then transforming them to take on pluripotent properties. Pluripotent cell differentiation done by manipulating specific genes back into the developing stage for expression similar to ESCs(1,8,15). Unfortunately, the propensity toward the formation of teratomas is also present once these cells genetically engineered (1). Furthermore, sex-specific differences also exist during the reprogramming process and may translate to differing potentials in future cellular development (15).

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Applications Once stem cells harvested, they then are grown in the laboratory (under in vitro environment) by being cultured within a dish containing nutrient broth. The in vitro process is repeated/propagated with replating/subculturing numerous times over several weeks to manufacture millions of ESCs. The proliferation of these healthy undifferentiated human embryonic stem cells (hESCs) for at least four weeks comprises an embryonic stem cell line. Freezing any batches of cells at a certain period can be done to preserve the cell population, which is subsequently packaged then sent elsewhere for more culturing (even cloning a whole organism) and utilized for other research experiments (8). Potential clinical utilization of stem cells toward the future treatment of autoimmune, neurodegenerative, inflammatory diseases, plus traumatic disorders has been studied in the laboratory and experimented in clinical trials on a limited basis (16). Stem cell therapy is aimed toward repair/replace any tissue unable to heal itself after being damaged or has become degenerated. Specific aims for research on stem cells directed toward alleviating pain or other symptoms of chronic disorders/illness or injury/trauma AND ultimately curing medical diseases. By growing, transplanting then directing healthy cells into the afflicted body parts, hopefully, additional new tissues will regenerate, including that stemness from native cells after stimulation for the final transformation (10).

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Basic methodology to grow Mesenchymal stem cells (MSCs) Adult MSC is the most versatile stem cell well tolerated in vivo with lesser teratoma conversion and histocompatibility issues. MSCs generally result in mesodermal differentiation and produce smooth and skeletal muscles, kidney cells (17,18,19,20,21,22,23,24, 83). Treatment/Therapeutic Strategies Since the1990s, the stem cell research field has acquired a tremendous amount of progress, especially with the potential of adult stem cells (ASCs) for further regeneration along with improved molding capability. Specifically speaking, the mesenchymal stem cells (MSCs) are proving to be a valuable cell source for management of medical conditions due to their multi-potency, the ability for widespread proliferation, along with relative ease of isolation/reproducible harvest. In effect, these stem cells can then be manipulated to directly differentiate into various cell lineages by targeting specific dysfunctional/distressed tissues. By using in vitro conditions,

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Journal Pre-proof laboratory scientists can culture large quantities of MSCs for delivery in vivo. Alternatively, cellular incorporation into an already engineered tissue can be done transplanting the final stage 4-5 cells completely differentiated cells inside the human body (1).

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Mesenchymal stem cells (MSCs) The initial characterization of MSCs appeared in the literature in the 1950s, first isolation attempted in the 1960s, then isolated a decade later in the 1970s in rats’ bone marrow yielding fibroblast-like cells. These in vitro cultured cells adhered to plastic and were capable of differentiation into various skeletal phenotypes: fibrous/fat-adipose tissue, muscle, tendon/ligament, cartilage, bone, marrow stroma along with other cell types (13,25,26). For more uniformity in human MSC isolation, 12 years ago, the International Society for Cell Therapy (ISCT) set out to refine three specific criteria for MSCs: plastic adherence in cell culture; cell-surface molecule expression; plus the capability to differentiate in vivo into chondroblasts, osteoblasts, and adipocytes (10). Furthermore, these potentially multi-potent cells also play a role in post-injury signals by secreting trophic and immunomodulatory factors. For this directing property, the name of “medicinal signaling cells” for MSCs has evolved. Along the same lines, research studies have demonstrated that all vascularized tissues contain MSC-like precursor cells or perivascular stem cells (PSCs). This population of cells contains an abundance of progenitor cells that can react quickly to begin the repair cascade upon tissue damage. However, in vitro conditions may not translate into in vivo results. Scientists are still debating whether the therapeutic effects of MSCs stem from differentiation and via cytokines and growth factors secretion (25).

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MSCs Sources Origins of MSCs within the body include placenta, umbilical cord, skin, adipose, tendon, muscle, synovium/fluid, epithelium, periosteum, bone marrow (BM), menstrual and peripheral blood amongst other bodily tissues (10,13,16,25,26,27). Interestingly, MSCs harvested from the synovium of patients with healthy joints are smaller than from subjects with rheumatoid or osteoarthritis (9). In clinical settings, the procurement of MSCs can be from one’s body-autologous (self-collection/injection) or via a donorallogenic (collected from another person then injected into the patient) (25). Autologous transplantation therapy uses the preferred feature of stem cells by avoidance of provoking a reaction from the recipient’s immune system, i.e., graft vs. host disease (GvHD) (16). The disadvantages of autologous cells include morbidity of the collection process and reduced differentiation capability with aging. On the other hand, allogenic cells are advantageous due to the ability to be mass-produced after being collected, along with being readily available “off the shelf” for use. However, the drawback of receiving stem cells from a donor is the potential of infectious disease transmission and transference of pathologic genetic material. Also, the risk of immunologic rejection of foreign cells by the recipient host exists as well as previously eluded to (25). Size/Age and Gender/Sex and Hormonal Factors Experimental studies were done in non-human primates, and mice/rat (murine) animal models have shown differences in the reduced ability of bodily tissue to

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regenerate with aging, equivalent comparison in the former species to humans between the third and fifth decade of age (28,29). Similarly, the “age” of donor cells makes a difference as far as both quality AND quality are concerned. More specifically, in a preclinical study, MSCs derived from umbilical cord blood can be cultured longer, multiply more, slower to grow older, and fight inflammation better than those isolated from the adult BM/adipose tissue. As for gender variation, pre-clinical research has shown that as compared to females, osteoblastic cell line in males showed a reduction in the ability to proliferate (28). In contrast, iPSCs from males tend to be epigenetically more stable in cell culture, as evidenced by yielding qualitatively more desirable stem cell lines. However, variability in cell expression profiles may not correlate with differences in eventual clinical function. Responses to regenerative medicine also differ between men and women, and even within women at various stages of their life, i.e., pre and/or postmenopausal states (29). Along the same lines, advanced age in women has a significant effect on the prevalence and cellularity of osteoblastic progenitor cells in the bone marrow (26,30,31). The result of post-menopausal osteoporosis from accelerated bone loss also supports the same outcome (26,30). More specifically, the capacity of BM-MSCs to proliferate and differentiate altered with aging, especially in females. A shift toward more adipocyte differentiation along with osteocyte plus MSC senescence contributes toward the reduction of both skeletal progenitor and terminally differentiated osteogenic cells, causing a decline of bone formation in elderly females (26). Thus, harvesting connective tissue progenitor cells from bone marrow aspirates from men results in quantitatively higher numbers in vitro for expression of bone phenotype after selective isolation, induction and differentiation into osteoblast-like cells (30). In addition, fibroblastic progenitor cells harvested from bone marrow can also be manipulated to differentiate into fat, fibrous/connective tissue, smooth muscle, and cartilage (26,28). Similarly, age was also found to be an essential variable in the chondrogenic potential of distal femoral bone marrow stem cells from males versus females, with the women yielding less compare to male donors as aging progress (32). However, alternative research on human synovial fat-derived MSCs has shown age not to be a significant factor between subjects in their mid-50’s as compared to those in their mid-80’s (33). Additionally, effective functional outcomes are affected more by the extent of the patient’s knee chondral defects (number and size) rather than recipient chronological age. Clinical results of knee joints post autologous inoculation of MSCs embedded in a hyaluronan-based scaffold were comparable in patients younger OR older than 45 years of age (28). On the other hand, confounding results were discovered by a group of researchers on MSCs harvested arthroscopically from the bone marrow of either the proximal humerus or distal femur in subjects over 20 years old. Some samples showed a difference in cell viability according to age, while others lacked any correlation between older versus younger patients, males or females (demonstrating no gender variability) (12). In other studies, differences have been shown to exist in stem cell characteristics due to (gender-specific) sex of donor cells AND hosts. As far back in ancient times beyond 2000 years earlier, the Greek philosopher Aristotle supposedly reported the existence of sexual dimorphism during the very early stages of embryonic development. More recently, within the past 20 years, similar sex differences have been observed in

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genetic expression between female versus male embryos. These observations indicate that even before the onset of hormonal exposure, genetic, cellular differing characteristics already exist between males and females (34,35). Scientists have shown that the activity of stem cells is gender-dependent thus can end up with significantly different results. Studies in Rhesus monkeys (2 years of age) have shown that mesenchymal stem cells harvested from females are more capable of neurogenesis than young male monkeys (16). This sex-specific finding has also been demonstrated in animal studies analyzing stem cell reaction to physiological stress. Activation of MSCs differs between murine XX and XY cells when exposed to hypoxic stress in an experimental environment. Regardless of recipient animals’ sex, XX cells behaved better than XY cells by producing more vascular growth factors to promote cell proliferation/regeneration and less necrosis factor, which affects inflammation/apoptosis or cell death. Additionally, the ability of muscle-derived stem cells (MDSCs) to regenerate also differs according to cell sex when analyzing a dystrophic (mdx) mouse model (muscle fibers lacking dystrophin protein). Although myogenic differentiation into dystrophin was similar by either cell sex in vitro, after quantification of muscle fibers, researchers have demonstrated the XX cells to ultimately yield a greater regenerative index (RI) than XY cells in vivo (34,36,37). In other words, when compared to male cells, regeneration of skeletal muscle is more efficient by female muscle-derived stem cells since they are not as sensitive to and thus are more capable of surviving against oxidative stress. Furthermore, female hosts receiving MDSCs also demonstrate the ability to regenerate significantly more than male recipients (35,36). Similarly, as compared to males, muscle progenitor cells harvested from females have been found to fare better in several tissue-engineered products. This may partly be due to females being able to handle foreign bodies (such as when carrying a fetus) with fewer complications from rejection (31). On the other hand, MDSCs from males tend to have the propensity for chondrogenic differentiation with improved potential toward cartilage regeneration (10). Furthermore, differences in the sex of cells plus sex of hosts also exist when studying muscle-derived stem cells treated in vitro with bone morphogenic protein (BMP4) resulting in osteogenic differentiation. Research has shown that male MDSCs transplanted into male recipients formed much more bone than female cells/hosts (35,36). Interestingly, other studies have shown that sex differences between stem cells affecting their ability to regenerate may not be dependent solely on male/female-specific hormones (34,35). On the other hand, research on 17 commonly utilized human ESC lines has found differences within same-sex stem cells due to their interaction with the hormonal milieu, both in vitro and in vivo (37). Variations of the stem and progenitor cells with their eventual reactions/cell cycle regulation, i.e., stimulation/division/proliferation/differentiation, are dependent on specific cell types under the influence of androgens in males and estrogens in females (16,37). For example, due to the influence of estrogen, female mice hematopoietic stem cells tend to divide much faster than males (16). Similarly, other scientists have found that male hormones cause inhibition vs. female hormones affecting stimulation of MSCs’ surface markers derived from synovial fat pad isolated from human knee joints (33). Since age/gender/sex of both donors and recipient hosts seem to have an effect on the ultimate results in animal models and human pre-clinical studies, one may

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Journal Pre-proof consider the balance of all factors by utilizing a mixture of autologous AND allogeneic stem cell transplantation to maximize functional outcomes while attempting to minimize inherent risks (16). Thirdly, BM‐MSCs harvested from individuals with increased BMI above 30 demonstrated reduced proliferation rates, increased senescence, and elevated expression of stress-related genes, along with significantly impaired osteogenic and decline in adipogenic differentiation. Therefore, consideration should be given to the weight/size of when choosing donor stem cell sources for tissue regenerative purposes (6).

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Tissue Engineering/Regenerative Medicine (TERM) for Musculoskeletal Health Basic Science and Clinical Applications One of the considerations in utilizing stem cells to treat orthopedic issues lies in the isolation process after harvesting. One must weigh/balance the specific sources available, procedure morbidity, and percent of cells ultimately produced. Bone marrow aspirate (BMA) was the initial site for MSC supply and also most commonly studied. Unfortunately, research has found that the collection of nucleated cells only yields relatively few MSCs (0.001-0.02%). Furthermore, the absolute cell number isolated drops off quite a bit as one becomes older. In addition, the rate of adverse reaction involved with BMA is 0.08%, which includes pain at the donor site, bleeding, and infection. Adipose tissue is another widely used donor source (ADSCs) obtained by liposuction since 2001. The original procedure was performed for cosmetic purposes by plastic surgeons, in the order of over 400,000 done every year in the U.S. With such an abundance of potential supply, procurement of adipose-derived stem cells is more readily available and much less invasive when compared to aspirating bone marrow. Additionally, the percentage of potential MSCs yield also far exceeds that from BMA, 17% of cells from the lipoaspirates, and up to 30-40% of crude stromal vascular fraction (SVF) post centrifugation. As far as quality, human ADSCs equate BMA in terms of chondrogenic and osteogenic capability, yet exceed the latter as far as being more stable morphologically/genetically. In comparison to BMA, ADSCs seem to be better at proliferation, along with retaining an extended period of differentiation potential in culture. Furthermore, ADSCs appear to also be better at protein secretion along with possession of enhanced immunogenic properties (25). As previously mentioned, the initial definition of human MSCs utilized in clinical studies was set by the International Society for Cytotherapy (ISC) in 2006. Three specific criteria must be met: plastic adherence upon maintenance while being cultured, expression of specific surface markers, and capability of differentiation into adipocytes, chondrocytes, and osteoblasts in vitro/Vivo (10,26). Other features of MSCs include the ability to migrate toward injured sites then releasing exosomes or trophic factors, which play a critical role in stimulating the surrounding milieu. They also affect cell signaling along with immune regulation for enhancement of angiogenesis, cytokine secretion, and differentiation. Ultimately, exosomes are responsible for supporting and promoting the inherent cellular regenerative capability in response to focal damage. Another mechanism through which MSCs contribute toward the reparative process involves immunomodulation. They help to make the local environment more resistant to autoimmune reactions by interacting with the body’s natural adaptive immune system

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Journal Pre-proof via anti-inflammatory and immunosuppressive effects. However, in order to have enough MSCs to exert their healing actions on damaged tissues, they must first be cultured in a medium then transported from the initial site of inoculation via a delivery scaffold. Once MSCs reach their intended destination, adherence, and integration into the target site is of paramount to successful mission completion (25). Figure 3:

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Several key components must occur in order to ensure the accomplishment of transplanted MSCs in its entirety: maintenance of cellular integrity, cellular attachment within the intended lesion, sufficient space for remodeling, and enough nutrition for cellular growth and differentiation. Adherence of MSCs occurs 3 hours post-injection, and if these crucial steps have not happened within 8 hours, fibroblastic differentiation will take place. The required sequence which MSCs must undergo within a certain timeframe could potentially affect protocols requiring longer joint immobilization postprocedure and, ultimately, clinical results. To enhance integration and incorporation of MSCs into targeted tissue, usage of a scaffolding agent and/or carrier vehicle has been investigated. Fetal bovine serum (FBS) has been commonly used as a culturing supplement but carries the added risk of infectious disease transmission or immunologic rejection (25). Platelet lysate (PL) or platelet-rich plasma (PRP) seems to behave similarly to FBS as far as efficacy without associated drawbacks. The reason lies in platelets containing more than 1500 protein-based growth factors along with being one of the initial responders to the injury site. As a result, upon using PRP as a scaffold adjunct, it has been proven to augment MSCs’ potential toward chondrogenic and osteogenic differentiation. Besides, this carrier system also imparts an anabolic influence on the extracellular matrix and the MSCs. As compared to controls, the combination of PRP and MSCs in vitro yielded ten times more chondrogenic markers (10,25). Alternatively, scientists have delved into using hyaluronic acid (HA) as an MSC adjunct in the culture medium, with improvement in growth kinetics, adhesion, and secretion of trophic factors in a dose-dependent fashion. Even though culturing media and scaffolding adjuncts appear to be safe and efficacious, any research involving cell culturing, enzymatic digestion of adipose tissue and/or treating cellular products with growth factors must approved by the FDA (13). Targeting specific orthopedic entities ATRAUMATIC Knee osteoarthritis/degenerative joint disease (OA/DJD) Osteoarthritis is an active, inflammatory, and progressive leading cause of the joint disorder, mainly involving hands or weight-bearing joints such as hip and knee, affecting women more than men, 30-40% of the elderly population over 65 years old, causing functional disability in the US. This degenerative joint disease affects at least 27million people, with prevalence increasing over time, projected to involve 67million by the year 2030, especially the obese, post-trauma, and retired athletes, also those with co-morbidities (endocrine/metabolic dysfunction), placing a tremendous financial strain

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upon society/economy (1,13,14,27,38,39,40). The monetary cost to our country is staggering, in the order of $128 billion (10). This eventual debilitating, degenerative condition affects more than just the chondral cartilage/articular surface. The subchondral bone and synovium are afflicted as well, resulting in stiffness and pain with secondary ligament laxity/muscle atrophy from bony deformity and disuse (13,27). Since articular cartilage is highly sensitive toward structural degradation along with being avascular, once this tissue is traumatized acutely or with repetitive usage upon chronic loading over time, it has the minimal intrinsic capacity to heal, resulting in ongoing chondral damage and ultimately ends in irreversible joint deterioration (14,38,41). This process ultimately ends in chondrocyte death and end-stage arthritis, which is accelerated by aging, especially since fewer cells are available combines with no blood vessels to start the reparative process (38). Currently, no medical cure exists to manage this disabling joint condition. Conventional surgical methods to treat articular defects include simple debridement, microfracture (bone marrow stimulation), OATS, or M/ACI (grafting procedure using scaffolds). Most of these procedures produce mainly fibrous tissue, which is different histologically and biomechanically from the original hyaline articular surface. None of these techniques has been found to recreate nascent cartilage or provide effective, long-lasting results to treat pain nor delay the arthritic progression (1,10,14,32,38). Patients who undergo total knee arthroplasty (TKA) to treat OA for an extended period definitively, as many as one in five people will still have painful symptoms or other issues post-surgery (14).

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Cartilage The natural characteristics of articular cartilage present itself as ideal for tissue engineering and regeneration. This connective tissue is lymphatic, aneural, and avascular. Cartilage is composed of only one type of cells-chondrocytes, along with ground substance/matrix (collagen/proteoglycans), and elastin fibers (14,27,38). With aging, the inherent properties of these constituents undergo biomechanical changes/structural alterations. Intra-articular chemokines and cytokines stimulate inflammation by producing and secreting proteases/destructive enzymes. The resultant effect damages the chondral surface, causing it to lose the ability for adaptation toward withstanding physical forces/stress load. In effect, an imbalance exists between the anabolic and inflammatory (mediators from synovium) catabolic pathways, causing matrix degradation and destroying homeostasis, ultimately ending in senescence (14,38). Therefore, in order for cartilage to undergo successful repair, novel surgical procedures/scientific methods must yield a large enough number of chondroprogenitors or phenotypically stable chondrocytes to enhance the integrative/restorative process. Thus utilizing MSCs as a source of replacement chondrocytes to treat articular lesions/OA is an attractive alternative to traditional procedures (38). Mesenchymal stem cells (MSCs) can be found in the dermis, bone marrow/aspirate, synovial membrane/fluid, articular cartilage, skeletal muscle, periosteum, adipose, and other adult connective tissues (38). The first isolation of adult MSCs from bone marrow was accomplished in 1999 by a research team led by Pittenger, showing the potential toward mesodermal/multilineage differentiation. They also possess the ability to adhere to the culture dish, allowing expansion in numbers without losing their multipotency status. In vitro, induction can be done for MSCs to produce tenocytes, myocytes, adipocytes,

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chondrocytes, and osteoblasts (38). After culturing, MSCs can differentiate into several types of specialized connective tissue, such as intervertebral disc, muscle, ligament, bone, and cartilage (10,26,38). Since MSCs have the capacity toward chondrogenic differentiation, OA has chosen as one of the main focuses of stem cell-based treatment. Manufacturing of tissue-engineered commercial products intended for cartilage repair therapy is on the rise. Near the end of 2016, MACI - autologous cultured chondrocytes harvested from an area of healthy cartilage expanded on scaffolds with bioabsorbable porcine collagen membrane then implanted over the focal damaged knee chondral surface; was approved by the FDA. The intended clinical population/usage indication is symptomatic full-thickness articular defects in knee joints of adult patients. Figure 4:

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To date, according to a review paper on different types of stem cell injections for knee OA in 6 clinical trials involving 155 patients, five randomized controlled trials (RCTs)-Level 2 and one nonRCT with Level 3-4 evidence, all studies appeared to contain bias (selection, performance, findings, analysis, and results). Selective stem cells harvested from autologous adipose tissue (uncultured), bone marrow, peripheral blood. Only one trial done without joint surgery, and the remaining investigators also included hyaluronic acid (HA) and PRP in addition to the injected MSCs. Patientreported outcome scores appeared to be positive at 24-48 months follow-up. Radiologic images also demonstrated positive findings. Two trials showed improvement of histologic findings and rates of arthroscopic healing. Although the authors reviewed cited favorable results in arthritic knees treated with stem cell injections, one must interpret this so-called clinical data with a critical eye since it seemed to contain mainly anecdotal evidence (27).

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Osteopenia/Osteoporosis Bone loss associated with aging (decline in bone mineral density-BMD) causing osteopenia and osteoporosis found in over 55 million people of the US population. As a result, more than 2 million fragility fractures occur yearly, resulting in associated morbidity/mortality plus costing our healthcare economy about 20 billion dollars! In order to combat this silent age-related thinning bone disorder, one must first understand the mechanism of BMD homeostasis. Bone is not static but undergoes continuous remodeling to maintain its structural integrity by regeneration of the whole skeleton every decade. This process is achieved by continuous osteoblastic formation keeping up with osteoclastic resorption, i.e., dynamic bone turnover. In other words, the terminal phenotypic differentiation of skeletal progenitors within the MSC reserve (osteoblasts) building bone, is balanced by the hematopoietic portion of mesenchymal stromal cellsMSCs (osteoclasts) destroying the bone. If a disturbance causes the former to slow down or the latter to speed up, then osteopenia or osteoporosis occurs due to net bone (primarily trabecular) loss. The pathology lies in the decline of skeletal stem cells (SSCs)-subset of MSCs in the bone marrow (BM) affecting females more so than males due to physiological sex steroid reduction associated with menopause. Treatment

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approaches aimed at either fighting catabolism/slowing down resorption and speeding up bone formation, anabolic therapy (26). As previously eluded to, bone marrow (BM) contains both hematopoietic and mesenchymal stromal cells. The latter are more in number consisting of perivascular cells possessing osteogenic progenitor properties, which aid in repair for maintaining bone mass. In mouse models, these multipotent self-cloning MSCs make up 1/1 million versus 1/100,000 (blood-line) BM cells in humans. This subset of BM cells consists of a heterogeneous population containing progenitor and terminally differentiated cell types. The former group includes SSCs, which are incredibly potent and dynamic progenitors. By utilizing clonal expansion, these self-renewing cells have been shown to possess the capacity for tri-lineage differentiation-bone, cartilage, fibrous but not fatty tissue. These SSCs tend to populate in long bone metaphyseal regions — their existence required for skeletal development, bone remodeling, and fracture healing. Bone strength is impaired, and skeletal volume reduced upon losing SSCs. Therefore, isolation of these skeletal stem cells for potential bone regeneration is being investigated extensively in animal and human studies (26). Animal models (mainly murine) are being utilized to investigate the feasibility of transplanted MSCs to induce new bone formation for the treatment of primary senile or secondary osteoporosis (hormonal or surgical). Thus far, positive results have shown in vivo, especially in studies where MSCs are modified to enhance osteogenic differentiation (26). In the past few years, close to 800 clinical trials utilizing MSCs for treating musculoskeletal conditions have approved by the FDA and registered on clinicaltrials.gov with ongoing patient recruitment.(10,26) A small percentage of these trials aimed toward bone healing for specific sites such as long bone fracture nonunion, tibial osteotomies, hip avascular necrosis (AVN). Since osteoporosis is a systemic condition affecting trabecular bone at various locations, it is more difficult to translate the efficacy of MSC delivery targeting just one single site into several bony areas (26).

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Sarcopenia Muscle atrophy is another age-related condition that is progressive, multifactorial, generalized, and sophisticated associated with decreased skeletal muscle mass, reduction in the number of muscle fibers along with alteration in muscle quality/physiology. The accompanied muscular strength loss causes the elderly to be frailer, putting them at higher risk of falling with resultant fractures, among other adverse events. Sarcopenia afflicts almost one-third of the older population causing physical disability, weakness, immobility, reduced life quality, functional decline, institutionalization, and even death. Available primary modalities of management for sarcopenia are nutrition and exercise. The former aims at making sure protein/amino/fatty acid intake is adequate for mass muscle maintenance. The latter focuses on slowing down the progression of muscular wasting/weakness by engaging in sufficient physical loading/resistance training for fall prevention and assist with daily activities/tasks to support independent living. Neither approach has been shown clinically to have a clear-cut advantage over the other in terms of addressing sarcopenia (42).

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Before scientists/clinicians can research on how to combat age-related changes resulting in sarcopenia, one must delve into the detailed pathophysiology. It appears that the reduction of muscle mass is much less evident than muscle strength loss. Studies in nonhuman primates and mice have shown that on the molecular level, defects exist in autophagy along with inadequate “clean up” of nonfunctional proteins. The resulting fast accumulation of oxidized along with cross-linked proteins causes cells to deteriorate and become dysfunctional. In addition, protective proteins against reactive oxygen species causing inflammation are not expressed secondary to mitochondria DNA mutations, along with a markedly decrease in the number of satellite (myogenic progenitor) cells; both may play a role in contributing to muscular atrophy, i.e., muscle fiber loss from programmed cell death. Other research has demonstrated that alteration of the microenvironmental niche rather than cellular changes themselves is responsible for the ability of myoblasts to differentiate and regenerate. When aging muscle cells implanted into a more youthful host, successful regeneration of skeletal muscle can occur, as long as the immune system is intact and functional (42). One other plausible factor contributing to sarcopenia in the elderly is neuronal remodeling, i.e., loss of motor neuron (MN) number and input to primarily fast-twitch (type IIB) muscle fibers. Compensatory hypertrophy of smaller MN occurs in skeletal muscle to innervate slower muscle fibers. This effect accounts for about one-tenth of the specific force decline found in rat experiments. Additionally, endocrine alterations in both males and females are also associated with muscular degeneration. Both sex-specific hormones, testosterone (primary anabolic hormone for protein synthesis) in men and estrogen in women stimulate satellite cells to proliferate for muscle regeneration. Estrogen has the additive action of modulation inflammation in response to injury as part of the healing cascade (42). Furthermore, the pancreatic hormone, insulin, plus other substances such as Vitamin D (VitD) and growth hormone (GH)/insulin-like growth factor (IGF-1) also play a role in age-related sarcopenia. Insulin exerts its anti-catabolic effects by decreasing protein degradation, along with an anabolic influence toward stimulating protein synthesis by improving amino acid absorption in the body. The hyperglycemic state in diabetics is associated with diffuse inflammation along with reduced muscle strength/mass. As for Vitamin D, supplementation older patients helps to increase muscle strength with a resultant decrease in fractures/falling. As for IGF-1, the level of which drops with aging is in parallel to GH decline secondary to impairment of the hypothalamic-pituitary axis. Since both of these chemicals are potent activators of cell proliferation, their diminutive presence contributes to muscle atrophic changes. Specifically, IGF-1 promotes satellite cell proliferation/protein synthesis, inhibits protein degradation, plus fights inflammation and scarring (42). Regenerative approaches for the treatment of sarcopenia need to expand beyond the in vitro expansion of stem cells then subsequent exogenous delivery into recipient hosts. Scientists must explore deeply into ways of duplicating the ideal microenvironmental niche conducive to fiber regeneration via satellite cells. Biologic scaffolds simulating mother nature’s healthy extracellular matrix (ECM) seem to stimulate myogenesis in animal models. The resultant effects on skeletal muscle have been demonstrated histologically, electromyographically, along with functional/strength improvement (42). Figure 5:

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Tendon Common locations of traumatic tendon entities include the knee extensor mechanism, Achilles, and shoulder (rotator cuff tears), especially with overhead athletic activity. Again, in addressing novel methods of management for soft tissue injuries, researchers/clinicians must hone in on replicating/replacing the basic cellular structure of tendons. Tendon is an extremely organized tissue made up of fascicles containing elongated, spindle-shaped cells, tenocytes that reside in the middle of tightly packed cross-linked fibers composed of Type I collagen (44). Due to their location at the ends of muscles and attachment to bone, tendons function to allow movements of joints. Due to their relative lack of vascular input, natural healing limited to post-trauma (10). Experimental studies in rabbits have shown that collagen gel constructs carrying BM-derived MSCs’ have limited success in repairing defects/gaps involving the Achilles tendon. Results were best at four weeks post-transplantation, with stiffness/strength reaching 200% versus controls (repaired by suture), but deteriorate over the next couple of months to values only about one-third that of healthy tendon integrity. Improvement of structural properties seen when a type I collagen sponge was seeded with gel and MSCs then stimulated mechanically in bioreactors, yielding stiffness/maximum force to failure closer to the native Achilles tendon. Besides their classic repopulating function for differentiation/growth, MSCs also secrete bioactive substances, which adds to their pleiotropic properties towards immunomodulation, chemotaxis, angiogenesis, antifibrosis, and anti-apoptosis effects. However, more research is needed to sort out which trophic characteristic(s) of MSCs would be most effective for tendon repair. The other challenge is to fabricate a fibrous biomaterial scaffold, which, after transplantation into the defective area, could eventually take on the biomechanical capability of normal healthy tendon. Furthermore, biological activity and long-term viability of these tissue-engineered constructs remain to investigate (44). Researchers in two human studies have investigated the use of stem cell injections for augmentation of rotator cuff repairs. One study involved 14 patients with no controls using bone marrow mononuclear cells (BMMCs) found the positive potential for better tendon regeneration. The other study was case-control with 45 patients utilizing MSCs injection in addition to standard surgical repair. Significant healing improvements were evident in terms of accelerated rate and retention of healed tendon integrity with a long-term follow-up a decade later, as demonstrated by ultrasound and magnetic resonance imaging modalities (45). Muscle The other type of soft tissue injury occurs in skeletal muscle, which comprises between 33-55% of all sports-related trauma. Gradation of injury severity includes contusion, strain, and laceration. Other indirect mechanisms causing muscular damage stem from overexertion or excessive intra-compartmental pressure post-trauma/swelling causing compartment syndrome, resulting in ischemia/neurologic dysfunction with

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subsequent muscle necrosis and death if not addressed on an urgent basis. Unfortunately, the pathophysiology of skeletal muscle injury portends a poor prognosis with associated morbidity. This is due to eventual dense fibrotic scar formation leading to muscle atrophy/painful joint contractures from reduced contractility/range of motion (ROM), prolonged rehab/recovery period, increased risk of repeat trauma, and inability to return to a prior level of baseline function or athletic performance. Traditional treatment of the injured muscle is RICE-rest, ice, compression, and elevation, along with local thermal application plus immobilization with regular ROM/functional rehab exercises. None of these modalities have an optimal effect on timely healing. Additional therapeutic measures in the form of medications such as steroids or NSAIDs can blunt the inflammatory response and may actually interfere with regeneration and even promote additional scarring (36). Muscle injuries present a challenge to surgeons upon attempted repair, though they usually heal (albeit with inferior tissue type) due to the reasonably abundant vascular supply (10). The series of events after the skeletal muscle is injured then ending in fibrosis preceded with myofiber degeneration, inflammation, and regeneration. The timeline for these steps occurs immediately post insult, next progress with the regenerative phase beginning within 24 hours then is completed after 3-5 days by the formation of new myofibers form myotubes. The final process is induced by cytokines stimulating previously dormant progenitor satellite cells (primary muscle stem cells) to differentiate into their destined myogenic lineage. Another, a better subpopulation of post-natal muscle-derived stem cells (MDSCs), which have isolated from muscle, has the potential for regeneration long term due to the number of population doublings (PD) up to 300 times before becoming senescent. This even exceeds embryonic stem cells (ESCs), which can only double themselves between 130-250. Furthermore, efficient transduction of MDSCs with antifibrotic and regenerative factors could assist in maximizing the healing of skeletal muscle post-injury. It appears that the origin of these cells comes from the endothelium of blood vessels. Ideally, it could utilize the MDSCs not only to repair the injury zone but also to replenish themselves by augmenting the local vascular supply (36). The majority of research on stem cell transplantation into impaired muscle is done on animal models (refer to the section on sex/gender differences). In a murine model, transplantation of MDSCs on the 4th day exhibited vascular ingrowth/angiogenesis at 7days, higher muscle strength after 14days, and less fibrotic tissue at 28 days. When transplanting on the 7th day, significantly less scarring observed. This animal study shows promise for stem cells having the potential to accelerate healing and minimize scar formation after muscle trauma (10). However, translation of basic bench scientific research to bedside has been limited with preliminary human trials using autologous MDSCs and direct implantation into injured skeletal muscle. The clinical focus has thus shifted toward the promotion of angiogenesis to stimulate satellite cells for healing enhancement through exercise and neuromuscular electrical stimulation (NMES) (36). Ligament On a cellular level, the composition of ligaments is similar to that of tendons, made up primarily of fibroblasts and extracellular matrix (ECM). Post-injury, the potential

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for ligament healing is partly dependent on its location, whether outside or within a joint. For the knee, extracapsular tissue such as the medial collateral ligament tends to heal better than the intrasynovial anterior cruciate ligament (ACL). Experimental trials using adipose-derived stem cells (ADSCs) have not been successful in terms of inducing them to convert into ligamentous tissue. Another study in rabbits using MSCs to augment ACL reconstruction with Achilles allograft at the tendon-bone tunnel interface yielded better osteointegration and biomechanical strength. Potentially this could be clinically applicable in humans for reconstructive purposes using donor tissue to help speed up graft incorporation (10). As of 5 years ago, a double-blinded study registered for investigating the combination of adult stem cells (ASCs) and hyaluronic acid (HA) injection versus HA alone as a control in patients up to 6 months post ACL reconstruction. Efficacy and safety will continue to be accessed/monitored over time in terms of interval imaging/side effects, respectively (10). Bone

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Bones compose the final component of the musculoskeletal system, which, when injured, can also be addressed using cell-based therapy. Fragility or stress fractures may benefit from stem cell treatment, especially in an already medically compromised/at-risk patient, to help enhance bony union. Using pigs as a model, adding ADSCs at two weeks post-surgery into the skeletal defect resulted in bone formation twice as fast plus doubled the volume of bone formed versus controls (10). Two human studies have been registered since 2013 to determine the effects of using MSCs for treating open tibia fractures and long bone nonunions to help enhance skeletal healing (10).

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Tissue engineering and regenerative medicine/stem-cell-based therapy are at the forefront of scientific investigation as an alternative, novel method of treating musculoskeletal disorders/orthopedic injuries. More progress has made in animal models and preclinical rather than human studies. Barriers to advancement in research lie in the US FDA governing body using its power to control clinical trials as an attempt to protect the public/patients until more relevant effective outcomes surface. A multitude of factors on both sides, donor and recipient hosts, will affect whether specific therapeutic stem cell combinations/modalities will succeed at their intended targets. Age, BMI, sex, gender, hormonal interactions, source, location, scaffolds, and local microenvironmental niche all play a role in influencing outcomes. Although numerous preclinical trials continue to show promise in terms of regeneration in several animals, a direct translation into patient care remains to be seen. Interpretation of available literature leaves much to be pondered in the foreseeable future, whether the “stem cell cure” is myth or magic, hope or hype? As a practicing clinician/orthopedic surgeon, I can only try to evaluate presented evidence with eyes wide open and an inquisitive mind. For certain conditions in practice, I can never cure with a knife, and I am excited at the positive prospect that stem cells may eventually bring to fruition for my patients and me…

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Acknowledgments: I would like to thank the following individuals for their prompt/expert assistance with this article: Kyla Petrie, MS2 in compiling the reference list; Aiswayra Pillai for illustrations & Mark Welborn for finalizing captions/figures.

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Journal Pre-proof 69. Savikj M, Ruby MA, Kostovski E, Iversen PO, Zierath JR, Krook A, Widegren U. Retained differentiation capacity of human skeletal muscle satellite cells from spinal cord-injured individuals. Physiol Rep. 2018 Jun;6(12):e13739. 70. Lian W, Hu X, Pan L, Han S, Cao C, Jia Z, Li M. Human primary CD34(+) cells transplantation for critical limb ischemia. J Clin Lab Anal. 2018Oct;32(8):e22569.

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71. Shahini A, Vydiam K, Choudhury D, Rajabian N, Nguyen T, Lei P, Andreadis ST. Efficient and high yield isolation of myoblasts from skeletal muscle. Stem Cell Res. 2018 Jul;30:122-129. 72. Tang N, Jiang S, Yang Y, Liu S, Ponnusamy M, Xin H, Yu T. Noncoding RNAs as therapeutic targets in atherosclerosis with diabetes mellitus. Cardiovasc Ther.2018 Aug;36(4):e12436. 73. Riccobono D, Nikovics K, François S, Favier AL, Jullien N, Schrock G,Scherthan H, Drouet M. First Insights Into the M2 Inflammatory Response After Adipose-Tissue-Derived Stem Cell Injections in Radiation-Injured Muscles. Health Phys. 2018 Jul;115(1):37-48. 74. Reddy A, Birur B, Shelton RC, Li L. Major Depressive Disorder Following Dermatomyositis: A Case Linking Depression with Inflammation. Psychopharmacol Bull. 2018 Mar 13;48(3):22-28. 75. Domenighetti AA, Mathewson MA, Pichika R, Sibley LA, Zhao L, Chambers HG, Lieber RL. Loss of myogenic potential and fusion capacity of muscle stem cells isolated from contractured muscle in children with cerebral palsy. Am J Physiol Cell Physiol. 2018 Aug 1;315(2):C247-C257. 76. Joshi J, Mahajan G, Kothapalli CR. Three-dimensional collagenous niche and azacytidine selectively promote time-dependent cardiomyogenesis from human bone marrowderived MSC spheroids. Biotechnol Bioeng. 2018 Aug;115(8):2013-2026. 77. Rønning SB, Pedersen ME, Berg RS, Kirkhus B, Rødbotten R. Vitamin K2 improves proliferation and migration of bovine skeletal muscle cells in vitro. PLoS One. 2018 Apr 4;13(4):e0195432. 78. Bajek A, Olkowska J, Walentowicz-Sadłecka M, Sadłecki P, Grabiec M,Porowińska D, Drewa T, Roszkowski K. Human Adipose-Derived and Amniotic Fluid-Derived Stem Cells: A Preliminary In Vitro Study Comparing Myogenic Differentiation Capability. Med Sci Monit. 2018 Mar 24;24:1733-174. 79. Thurner M, Asim F, Garczarczyk-Asim D, Janke K, Deutsch M, Margreiter E, Troppmair J, Marksteiner R. Development of an in vitro potency assay for human skeletal muscle derived cells. PLoS One. 2018 Mar 22;13(3):e0194561. 80. Čamernik K, Barlič A, Drobnič M, Marc J, Jeras M, Zupan J. Mesenchymal Stem Cells in the Musculoskeletal System: From Animal Models to Human Tissue Regeneration? Stem Cell Rev Rep. 2018 Jun;14(3):346-369. 81. Ebrahimi MJ, Aliaghaei A, Boroujeni ME, Khodagholi F, Meftahi G, Abdollahifar MA, Ahmadi H, Danyali S, Daftari M, Sadeghi Y. Human Umbilical Cord Matrix Stem Cells Reverse Oxidative Stress-Induced Cell Death and Ameliorate Motor Function and Striatal Atrophy in Rat Model of Huntington Disease. Neurotox Res. 2018 Aug;34(2):273-284.

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82. Turner MC, Player DJ, Martin NRW, Akam EC, Lewis MP. The effect of chronic high insulin exposure upon metabolic and myogenic markers in C2C12 skeletal muscle cells and myo tubes. J Cell Biochem. 2018 Jul;119(7):5686-5695. 83. Reddy AP, Ravichandran J, Carkaci-Salli N. Neural regeneration therapies for Alzheimer's and Parkinson's disease-related disorders. Biochim Biophys Acta Mol Basis Dis. 2019 Jul 2:165506.

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Journal Pre-proof Legend Figure 1: The origin of these pluripotent cells (potential to differentiate into multiple cell types) derived from the inner cell mass of 4-5 days-old embryos (blastocysts-hollow microscopic sphere of cells). This process is harvested before uterine implantation, and these cells are then potentially immortalized to self-replicate/renew indefinitely while remaining in an undifferentiated state. Since ESCs are isolated from multiple embryos, specific cell lines can thus be delineated to showcase unique features of gene expression post differentiation eventually (1, 12, 11, and 83).

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Figure 2: Mesenchymal stem cells (MSCs) originating from bone marrow (BM). Mesenchymal stem cells originally part of one of the three distinct layers of embryo (Ectoderm, mesoderm, and endoderm) MSCs are multipotent mesoderm origin can differentiate in cartilage, muscles, and bones in the in vitro conditions (1,12). Human MSCs, AKA connective tissue progenitor cells for the mesoderm lineage, consist of stromal, nonhematopoietic cells with the potential for differentiation into dermis, fat, muscle, ligament, tendon, cartilage, and bone (12,13).

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Figure 3: MSCs differentiation and pluripotency: MSCs differentiation depends on cell characteristics, and usage and figure explain the multidimensional usage of cells to regenerate cells for grafting studies.

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Figure 4: The process of chondrocyte grafting. Pre-clinical investigation has shown promise with MSC injection for OA therapy in several animal models: meniscal repair/regeneration in rat/rabbit/goat/sheep; cartilage repair/protection/arthritis prevention in Guinea pig (with added HA), rabbit and donkey/mouse/rat, respectively; along with synovial fluid improvement in horses (13).

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Figure 5: Tendinopathy. The paucity of information exists in the literature regarding stem cell usage for degenerating tendons. Only one study has found in the national database as of 3 years ago. More studies have done on veterinary equine athletes such as race/show horses. Bone marrow-derived stem cells postexpansion in culture after which injected into a horse’s limb tendon similar to human’s Achilles showed improvement of histological/biomechanical characteristics in a randomized study (N=12). The host tendons had restoration of stiffness and reduced rupture rate by approximately 50%. How these results may fare out in humans has yet to be determined (43). TRAUMA/INJURY. In 2005, 10% of the US population missed work due to trauma, resulting in over 72 million lost days at their job (7). As of 6 years ago, more than 60% of patient who seeks medical care for injuries consist of musculoskeletal ailments. Almost one-third of these injuries are due to soft tissue trauma such as ligament sprains/tendon strains or ruptures. The financial impact on the healthcare system is tremendous, costing over $30 billion yearly. Recreational sports-related injuries in the “weekend warriors” exceed that of vocational accidents in the workplace (44).

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Tables1: Biomarkers in stem cell research in musculo-skeletal disorders. Tissue/cells Blood cells nexgene seq

TPM2 gene

Nematin myopathy in iPSC from PMBC cells Dermal fibroblast stem cells

DMD gene

CFTR gene

Gut muscles

EMT cells muscle

Epithelial-MSCs, bladder cancer and marker, SSH Serum biomarker Osteoarthritis

DMD/Dp427isoform

Proteomics markers for DMD

MSCs

MSCs isolation from different human tissues (AT-MSCs, SK-MSCs, BMMSCs) Biomarkers for spinal muscular atrophy(SMA)

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re

lP

na

SMA

hiPSC and hESCs comparison in sinoatrial differentiation

Muscle biopsy

Skeletal muscles from cancer chemotherapy patients.

Myo miRNA

miRNA in Amyotrophic lateral sclerosis(ALS) FAPs role in DMD.

hESC –SMN1 mutation

Fibroadipogenic progenitors(FAPs)

Jo

HIPS(iPSCs)

Fibro-Adipogenic progenitor (FAPs)

of

MSCs

iPSC neurons from NSC (mouse model) Bone marrow derived

ur

SMARD1 gene

Outcome Metabolic myopathies due to enzyme deficiency.(46) NEM4 mutation retain in iPSC differentiation.(47) iPSC expresses the reprogramming markers OCT4, SOX2, LIN29, LKF4 and L-myc.(48) Expresses markers Lewis X, CXCR4 and integrin β.(49) Distinct cell surface and angiogenic markers after transplant.(50) Co-localization of CFTR and Choli acetyltransferase.(51) Sonic hedgehog increased in cancer cell dependence.(52) Creatinine kinase muscle (CK-MM and Aldolase A(AldoA) markers beside pain and anatomy.(53) Fiber contraction, cell signaling, ion homeostasis, cell stress, cell metabolism and immune response. (54) Quality control analysis of MSCs cellular and biomarkers.(55)

ro

Genes/miRNA/Biomarker CPT2 gene

Spinal muscular atrophy (SMA)due to SMN1 mutation differentiated in myogenic cells Adult skeletal progenitor stem cells helps in muscle repair.

Phosphorylated neuro filament heavy chain(pNF-H) elevated in plasma of infant having SMA.(56) The marker comparison of human induced pluripotent stem cells and human embryonic stem cells.(57) Glut4,CytO-C, COXIV,SDHA and VDAC most importantly altered in exercise induced energy cascade.(58) miR-1,miR 133-a and miR133-b established biomarkers myo miRNA (59) IL-B1 and IL-4 altered FAP s cells and polarized cells results in accumulation of inflammatory cytokine induced fat accumulation.(60) Cells found be deficient in cholinergic calcium, signaling , glycolysis and Oxphos biological mechanism.(61) OSR1 higher expression activate FAPs.(62)

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Journal Pre-proof MuSC differentiation.

Adipose mesenchymal stem cells(ADSCs) biomarker

Differentiation ADSC in rat differentiated insulin induced stem cells. Liver injury due dystrophinopathy.

MyD88 marker in DMD

Role of MyD88 in DMD pathology

DMD, BeMD, LGMD

Bone defect in neuromuscular disorders. Vitamin D receptor (VDR) in postmenopausal osteoporosis (PMO)

Post-menopausal osteoporosis(PMO)

Spinal cord injury and regenerative plasticity of satellite cells.

CD34+ cells

Grafting human primary CD34+ cells in limb ischemia.

SkMSCs

Skeletal muscle myoblast high yield and generation. Multiple long noncoding RNA in Atherosclerosis and diabetes mellitus.

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Skeletal satellite cells

Noncoding RNA

PAX7 downregulation increases ECFP expression and differentiate MUSC(63) Adipogenic markers PRDM16,UCP1 andPGC1α and myogenic markers MyoD and MyoG regulated with miRNA-499.(64) Ration of creatinine kinase and Alanine aminotransferase a biomarker in acute liver injury(65) Activation of MyD88 reduce response to inflammatory pathways.(66) High level of Leptin may interfere bone formation and leads to bone defect.(67) VDR dysfunction leads to osteoporosis. Genetic polymorphism rs1544410G/G linked to PMO.(68) Myogenic differentiation factor1 high expression during injury. AKT signaling, rapamycin and forkhead O complex altered in spinal cord injury.(69) Increase vascular density and higher expression of VEGF and bFGF higher in recovery.(71) Matrigel from chicken serum and bFGF increases α7-Integrin,MyoD,and Desmin.(71) lncRNA>200, miRNA 17-25, circ RNA , piRNA 26-32 may be potential biomarker for therapeutic targets in DM and ACS.(72) ADSCs from radiation tissue biopsies have higher expression of inflammatory markersM1 (IL1-β and IL-6) and M2(IL10 and TGF-β)(73) Inflammatory markers altered in MDD patients with Dermatomyositis.(74) CP differentiate muscles satellite cell loose the potential myo tube and myogenic markers.(75) WNT signaling and Azacytidine-5 is important for cardio myocytes development and maturity.(76) Vitamin K improve in vitro myogenesis.(77) OCT4, SOX2, CD34, CD44, CD45 and CD 90 assessed in both differentiation and found that ADSCs ( Adipose) derived has better myogenic differentiation.(78) Acetylcholinesterase assay for CD+ cells may produce multinucleated myo tubule may result in mature skeletal muscle cells.(79)

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MuSCs

Adipose tissue derived muscles cell from radiation cutaneous exposure syndrome.(CD80/CD206)

Major depressive disorder(MDD) Cerebral Palsy(CP)

MDD linked to Dermatomyositis.

BM MSCs

3D cardiomyogenesis.

SMSCs hADSCs and hAFSCs

Skeletal muscles in aging. Comparison of human amniotic fluid derived and human adipose tissue derived stem cells.

hSMDCs

In vitro assay for stem cell potency.

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ADSCs

CP muscle cells differentiation.

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Regenerative capacity of MSCs from different areas. Umbilical cord generated neurons in Huntington disease.

UMSCs

hMSCs

Chronic insulin treatment.

Assessment of various areas MSCs.(80) In the rat model of HDUMSCs grafting in striatum survive neurons and improve muscle activity.(81) Chronic insulin treatment to skeletal muscle differentiation improve glucose uptake and alter AKT signaling via reduce phosphorylation.(82)

NCT0 2881 047* NCT0 2235 844¤ NCT0 0010 335¤ NCT0 2131 077¤ NCT0

ro

-p

High concentration of Allo-ASC

Cerebral Palsy, Spastic Subjects With Severe, Refractory Sclerotic Skin Changes

na

Lateral Epicondylitis

Institut de Terapia Regenerativa Tissular Seoul National University Hospital|Korea Health Industry Development Institute Tehran University of Medical Sciences|Hormozgan University of Medical Sciences

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mesenchymal stem cells

ur

NCT0 3795 974*

Sponsor/Collaborators

MNC

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NCT0 3449 082*

Interventions

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Clinical trials in musculodisorder Past and current NCT Num ber Conditions NCT0 3454 Patellar 737* Tendinopathy

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Table 2: Clinical trials in Musculoskeletal disorders.

Age * only male subjects

Enrollment

18 Years to 48 Years *

20

19 Years and older

30

4 Years to 14 Years

108

18 Years and older

8

28 Years to 31 Years *

1

Duchenne's Muscular Dystrophy Systemic SclerosisJuvenile Rheumatoid Arthritis

Umbilical Cord Mesenchymal Stem Cells

Abramson Cancer Center of the University of Pennsylvania Allergy and Asthma Consultants, Wichita, Kansas Aidan Foundation

Stem Cell Transplantation CD34

Fred Hutchinson Cancer Research Center

2 Years to 18 Years

6

Tennis Elbow Compartment

ALLO-ASC-TI Magellan®

Anterogen Co., Ltd. Arteriocyte, Inc.

19 Years to 90 Years 18 Years to 65

30 5

ablative fractional laser

31

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Sporadic Inclusion Body Myositis Mitochondrial Diseases|Pearson Syndrome Inherited Cardiac Arrythmias, Myotonic Dystrophy RSD (Reflex Sympathetic Dystrophy)Fibromy algia

Blood-flow restricted training

University of Southern Denmark|Odense University Hospital

35 Years and older

22

CD34+ cells enriched with MNV-BLD

Minovia Therapeutics Ltd.

Child, Adult,

7

Muscular Dystrophies

Johns Hopkins University (NHLBI)

18 Years to 85 Years

100

16 Years and older

100

5 Years to 15 Years

45

18 Years to 80 Years

90

18 Years to 90 Years

200

18 Years to 95 Years

80

4 Years to 25 Years

20

18 Years to 60 Years

16

18 Years and older

200

40 Years to 75 Years

246

NCT0 3942 445¥

Artery Disease|Muscle Disorder

Gastrocnemius muscle biopsy

NCT0 3279 796¥ NCT0 2985 424¥ NCT0 3067 831¶ NCT0 3477 942¶ NCT0 3390 920¶ NCT0 3752 827¶

Rotator Cuff Tear|Lateral Epicondylitis Polymyalgia Rheumatica|Giant Cell Arteritis Duchenne Muscular Dystrophy Musculoskeletal Pain,Cartilage Degeneration OsteoarthritisSport s Injury, Neuropathy Rotator Cuff Tear|Rotator Cuff Tendinitis

NCT0 2611 089¶ NCT0 3156

Radial Dysplasia Cardiovascular Diseases

Healeon Medical Inc

Assiut University Institut National de la Santé Et de la Recherche Médicale, France Second Affiliated Hospital, School of Medicine, Zhejiang University Svendborg Hospital|Odense University Hospital

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Myopathy

Lipoaspiration Sildenafil, Mesenchymal stem cell transplantation

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NCT0 2987 855£ NCT0 3633 565¥

Years

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NCT0 2413 450£

Syndrome

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1837 264¤ NCT0 2317 094¤ NCT0 3384 420£

na

Autologous adiposederived MSC

Jo

ur

18F-FDG PET/CT

Stem Cells Autologous Mesenchymal Stem Cells

Amniotic Adipose Derived Regenerative Cells, Corticosteroid

Tissue biopsy Blood volume collected

Stem Cells Arabia University Hospitals Cleveland Medical Center

R3 Stem Cell

InGeneron, Inc. King's College London,Royal Free Hospital NHS Foundation Trust University of Florida|Florida Space

up to 18 Years 21 Years to 50 Years

30 20

32

Journal Pre-proof Institute

NCT0 0006 055Ø NCT0 2869 061Ø NCT0 2241 928§ NCT0 2245 711§ NCT0 2241 434§

Stem Cell Umbilical Cord Based Allogenic Mesenchymal Stem Cell ALLO-ASC(allogeneic adipose derived mesenchymal stem cell Autologous Mesenchymal Stem Cells

ur

Lateral Epicondylitis Achilles Tendinitis,Achilles Degeneration Rheumatoid Arthritis, Systemic Lupus Erythematosus

Jo

NCT0 1856 140Ø NCT0 2064 062Ø

Bladder Neck Obstruction Muscular Dystrophy Limb Girdle Muscular Dystrophy Duchenne Muscular Dystrophy

Northwestern University

15 Years to 65 Years

9

Chaitanya Hospital, Pune

4 Years to 20 Years

30

Acibadem University

7 Years to 20 Years *

10

Shenzhen Beike BioTechnology Co., Ltd

5 Years to 12 Years

15

Chaitanya Hospital, Pune

6 Years to 25 Years

25

University of Gaziantep, Gaziantep Deva Hospital,

8 Years to 14 Years *

10

Seoul National University Hospital

19 Years to 90 Years

12

University College, London

18 Years to 70 Years

10

1 Year to 55 Years

10

40 Years to 70 Years *

12

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Duchenne Muscular Dystrophy

7

-p

NCT0 2484 560Ø

NCT0 1834 040Ø NCT0 2285 673Ø NCT0 1610 440Ø

lP

NCT0 1834 066Ø

Myasthenia Gravis Muscular Dystrophy|Duchen ne Muscular Dystrophy Duchenne Muscular Dystrophy Duchenne Muscular Dystrophy Muscular Dystrophy|Duchen ne Muscular Dystrophy,

Hematopoietic Stem Cell Transplantation Intralesional/ Intravenous of Autologous Stem cells. Umbilical Cord Mesenchymal Stem Cell human umbilical cord mesenchymal stem cells

16 Years to 65 Years

re

MYOPATHY

Northwestern University

ro

Hematopoietic stem cell transplantation, Mesna

na

257¶ NCT0 0278 564µ NCT0 0424 489µ

anti-thymocyte globulin, Stem Cell Transplantation Liposuction, ADRC isolation

Fairview University Medical Center|Office of Rare Diseases (ORD) Central Clinical Hospital , Moscow State Medical University

Stem Cell

Neurogen Brain and Spine Institute

6 Months to 60 Years

0

Stem Cell

Neurogen Brain and Spine Institute

15 Years to 60 Years

0

Stem Cell

Neurogen Brain and Spine Institute

3 Years to 25 Years

0

33

Journal Pre-proof NCT0 2050 776§

Limb Girdle Muscular Dystrophy

Autologous bone marrow mononuclear cell transplantation

Neurogen Brain and Spine Institute

0

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Status:Active not recruting= *, Com plete d=¤ , Enrolling by invitation=£, Not recruiting=¥, Recr uting =¶,

15 Years to 60 Years

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Term inate d =µ unknown status=Ø, With draw n=§

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5