**2.2 Adipose tissue grafts**

Stem cell therapy uses non-embryonic adult stem cells described as multipotent stem cells, and in the clinical setting refers to therapy with mesenchymal (often from adipose tissue) and hematopoietic stem cells (often from bone marrow aspirate) [3]. These cells are present in a variety of tissues (adipocytes, chondrocytes, myocytes) and are thought to play a role in immune modulation [3]. The most common source of mesenchymal stem cells (MSC) are found in adipose tissue, first discovered in 1964 by Rodell [2]. There are approximately 500 to 2500× times more MSC's when compared to bone marrow [3]. Adipose derived stem cells (ASC's) are the most promising stem cells identified in humans, since adipose tissue is easily obtained in large quantities with small donor site discomfort [4]. Sites of harvest include the abdomen, upper arm, thigh, and trochanteric fat deposits. Common mechanisms to obtain fat include liposuction or lipectomy, followed by homogenization and enzymatic digestions. Traditional cosmetic liposuction can remove large volume (>4 kg) or small volume (<4 kg) adipose tissue, however for purposes of adipose tissue grafting only 100–200 mL may be needed [9]. The resultant material is then centrifuged. Each gram of adipose tissue yields 5 × 10^3 stem cells, significantly greater than bone marrow [4]. It is important to note that stem cell harvesting is more invasive that a simple blood draw for PRP, and could thus lead to an increase risk for infection or complication for patients undergoing MSC harvesting [10]. If performed under local or tumescent anesthesia, there is minimal to no recovery time. To date, hundreds of trials are listed on the United States National Institutes of Health website (NIH) for the use of ASC's. Examples of applications include soft tissue regeneration, skeletal tissue repair, myocardial infarction, immune disorders such as lupus, multiple sclerosis, Crohn's disease, diabetes.

#### **2.3 Bone marrow aspirate concentrate**

Bone marrow aspiration is a procedure in which bone marrow is collected, usually from the pelvic iliac crest [11]. The procedure is very similar to PRP, in which the product is centrifuged. The final product is called bone marrow aspirate concentrate, or BMAC, which contains mostly hematopoetic stem cells (HCC's), and a much smaller concentration of MSCs [3]. Like MSC's, HSC's also contain growth factors and immunomodulating enzymes [11]. Unlike adipose aspiration, the concentration of MSC's in bone marrow dramatically decreases with age. Like ASCs, BMAC is also used to treat various conditions affecting tendons, ligaments, and musculoskeletal injuries. Approximately 60 mL of aspirate can produce 10 mL of BMAC after centrifuge [11]. Again, efficacy is determined by location of injection as well as extent of tissue injury. Sites include shoulders, knees, hips, various tendons, and sometimes spinal facet joints. Current literature demonstrates benefit in utilizing BMAC as an adjunct in cartilage healing, faster time to bony union, and lower rates of tendon re-rupture [11].

#### **2.4 Exosomes**

Exosomes are endocytic vesicles released by various cells including T-cells, B-cells, reticulocytes, mast cells, platelets, tumor cells as well as MSCs [12]. They are membrane-enclosed particles surrounded by a phospholipid layer and are enriched with micro-RNAs (miRNAs) which are believed to regulate gene expression in a post-transcriptional matter and, by that matter, play a role in tissue repair and regeneration [13, 14]. They are defined as nanosized membrane vesicles with a diameter of 30–100 nm that originate from multivesicular bodies (MVB's) and are released by cells into extracellular environment (**Figure 3**) [12]. They are cholesterolrich phospholipid vesicles. There are multiple contents that are found in exosomes including cytokines, proteins, lipids, mRNAs, miRNAs and ribosomal RNAs [15, 16]. Current recommendations for extraction of exosomes suggest ultracentrifugation at high speeds which removes cells and microvesicles. It is time consuming, labor intensive, and has therefore lead to commercially available kits, such as Exoquick ©, Invitrogen ©, and Exo-Spin ©, which reliably reduce operating time to 2 hours. Exosomes ultimately have the capacity to execute specific targeted therapy due to their ability to envelope a wide range of specific contents, including lipids, RNA's, and specific protein-signaling molecules [17]. This makes them a promising tool in nanomedicine; however, like PRP and MSC's, classification and purification needs to be standardized to ensure appropriate randomized and multicenter studies.

#### **2.5 Mechanism of action and biology**

The mechanism by which these injections treat pain is still unknown and remains mainly theoretical [15]. Platelets, also called thrombocytes, contain several secretory

**23**

*Regenerative Medicine*

**Figure 4.**

*Signal transduction pathways.*

*DOI: http://dx.doi.org/10.5772/intechopen.93717*

granules crucial to platelet function. Platelets are primarily responsible for aggregation and contribute to hemostasis through adhesion, activation, and aggregation. In recent years, research has demonstrated that platelets not only have a function in hemostasis, but also carry an abundance of growth factors and cytokines that affect inflammation, stem cell migration, and proliferation [8]. Upon activation of platelets in PRP, important growth factors, namely endothelial growth factor, fibroblast growth factor, platelet derived growth factor, epidermal growth factor, hepatocyte growth factor, matrix metalloproteinases, and interleukin 8 are released and modify

the local tissue through a complex cell signaling cascade (**Figure 4**) [6].

neurites and promote neurogenesis [22, 23].

There is increasing surfacing evidence suggesting that MSC's secrete functional paracrine factors which promote tissue regeneration, protection and repair [18–21]. Recent studies revealed that factors are released through particles known as exosomes. There is now strong belief that these exosomes secreted by MSCs are responsible for the tissue regeneration and repair rather that the ability of MSCs to trans-differentiate. They are suggested as central mediators of intercellular communication by transferring proteins, mRNAs and miRNAs to adjacent cells leading to coordinative function in organisms. Those, depending on the medium in which they are collected can halt breast cancer metastasis, regulate angiogenesis, remodel

Exosomes aggressively participate in the inhibition of the inflammation process. This known property of exosomes is now being considered as a novel therapeutic approach to many disease processes affecting different organ systems. Indeed, the benefits of the anti-inflammatory properties of exosomes has not only been demonstrated in various painful conditions, but also in cardiovascular and pulmonary diseases as well. MSCs-secreting exosomes lead to a reduction of white blood cell count and reduced inflammation in hearts after ischemia-reperfusion injury, which in turn decrease infarct size and enhance cardiac function. Protective properties have also been shown in the lungs for relieving symptoms of pulmonary hypertension [24, 25]. MSCs are adult multipotent cells with the ability to self-renew and differentiate into mesenchymal lineage such as osteoblasts, chondrocytes and

**Figure 3.** *Multivesicular bodies (MVB's) releasing exosomes.*

*Pain Management - Practices, Novel Therapies and Bioactives*

**2.4 Exosomes**

product is centrifuged. The final product is called bone marrow aspirate concentrate, or BMAC, which contains mostly hematopoetic stem cells (HCC's), and a much smaller concentration of MSCs [3]. Like MSC's, HSC's also contain growth factors and immunomodulating enzymes [11]. Unlike adipose aspiration, the concentration of MSC's in bone marrow dramatically decreases with age. Like ASCs, BMAC is also used to treat various conditions affecting tendons, ligaments, and musculoskeletal injuries. Approximately 60 mL of aspirate can produce 10 mL of BMAC after centrifuge [11]. Again, efficacy is determined by location of injection as well as extent of tissue injury. Sites include shoulders, knees, hips, various tendons, and sometimes spinal facet joints. Current literature demonstrates benefit in utilizing BMAC as an adjunct in cartilage healing, faster time to bony union, and lower rates of tendon re-rupture [11].

Exosomes are endocytic vesicles released by various cells including T-cells, B-cells, reticulocytes, mast cells, platelets, tumor cells as well as MSCs [12]. They are membrane-enclosed particles surrounded by a phospholipid layer and are enriched with micro-RNAs (miRNAs) which are believed to regulate gene expression in a post-transcriptional matter and, by that matter, play a role in tissue repair and regeneration [13, 14]. They are defined as nanosized membrane vesicles with a diameter of 30–100 nm that originate from multivesicular bodies (MVB's) and are released by cells into extracellular environment (**Figure 3**) [12]. They are cholesterolrich phospholipid vesicles. There are multiple contents that are found in exosomes including cytokines, proteins, lipids, mRNAs, miRNAs and ribosomal RNAs [15, 16]. Current recommendations for extraction of exosomes suggest ultracentrifugation at high speeds which removes cells and microvesicles. It is time consuming, labor intensive, and has therefore lead to commercially available kits, such as Exoquick ©, Invitrogen ©, and Exo-Spin ©, which reliably reduce operating time to 2 hours. Exosomes ultimately have the capacity to execute specific targeted therapy due to their ability to envelope a wide range of specific contents, including lipids, RNA's, and specific protein-signaling molecules [17]. This makes them a promising tool in nanomedicine; however, like PRP and MSC's, classification and purification needs to

be standardized to ensure appropriate randomized and multicenter studies.

The mechanism by which these injections treat pain is still unknown and remains mainly theoretical [15]. Platelets, also called thrombocytes, contain several secretory

**2.5 Mechanism of action and biology**

*Multivesicular bodies (MVB's) releasing exosomes.*

**22**

**Figure 3.**

**Figure 4.** *Signal transduction pathways.*

granules crucial to platelet function. Platelets are primarily responsible for aggregation and contribute to hemostasis through adhesion, activation, and aggregation. In recent years, research has demonstrated that platelets not only have a function in hemostasis, but also carry an abundance of growth factors and cytokines that affect inflammation, stem cell migration, and proliferation [8]. Upon activation of platelets in PRP, important growth factors, namely endothelial growth factor, fibroblast growth factor, platelet derived growth factor, epidermal growth factor, hepatocyte growth factor, matrix metalloproteinases, and interleukin 8 are released and modify the local tissue through a complex cell signaling cascade (**Figure 4**) [6].

There is increasing surfacing evidence suggesting that MSC's secrete functional paracrine factors which promote tissue regeneration, protection and repair [18–21]. Recent studies revealed that factors are released through particles known as exosomes. There is now strong belief that these exosomes secreted by MSCs are responsible for the tissue regeneration and repair rather that the ability of MSCs to trans-differentiate. They are suggested as central mediators of intercellular communication by transferring proteins, mRNAs and miRNAs to adjacent cells leading to coordinative function in organisms. Those, depending on the medium in which they are collected can halt breast cancer metastasis, regulate angiogenesis, remodel neurites and promote neurogenesis [22, 23].

Exosomes aggressively participate in the inhibition of the inflammation process. This known property of exosomes is now being considered as a novel therapeutic approach to many disease processes affecting different organ systems. Indeed, the benefits of the anti-inflammatory properties of exosomes has not only been demonstrated in various painful conditions, but also in cardiovascular and pulmonary diseases as well. MSCs-secreting exosomes lead to a reduction of white blood cell count and reduced inflammation in hearts after ischemia-reperfusion injury, which in turn decrease infarct size and enhance cardiac function. Protective properties have also been shown in the lungs for relieving symptoms of pulmonary hypertension [24, 25]. MSCs are adult multipotent cells with the ability to self-renew and differentiate into mesenchymal lineage such as osteoblasts, chondrocytes and

adipocytes [26]. MSCs were originally isolated from bone marrow, they have been isolated from other adult tissues such as adipose tissue, dental pulp, placenta, amniotic fluid, umbilical cord blood and Wharton's jelly, and even in the brain [27]. The MSCs differentiation potential for tissue repair has been studied extensively but the pattern of MSC mediated regeneration is now shifting toward secretomebased paracrine activity. The matter by which miRNAs play an essential role in physiological and pathological conditions is by regulating gene expression at the post-transcription level [28]. The pre-miRNA goes through an extensive biological process prior to maturation but an exosome can contain miRNA of different maturation stages and their release is a controlled process dependent on the source and developmental stage of derived cells rather than a random process [12]. It has been suggested that exosomes released by MSCs contain miRNA that control the microenvironment in the resident niches through a balance between proliferation and differentiation [29]. Additionally, tissue-specific responses have been described for exosomes isolated from different sources. For example, adipose tissue-derived exosomes seem to be more effective in halting the central nervous system degeneration caused by Alzheimer's disease when compared to bone marrow derived MSCs-derived exosomes [30]. But neurite outgrowth seems to be more responsive to exosomes released by menstrual fluid derived MSCs when compared to umbilical cord, chorion and bone marrow. Evidence of neurite outgrowth has also been shown in a middle cerebral artery occlusion model. MSCs exposed to ischemic cerebral extracts secreted exosomes containing mi-RNA that were transferred to neurons and astrocytes via exosomes and promoted neurite outgrowth and functional recovery. The same authors reported the use of cell free MSC-generated exosomes administered intravenously in a subject that had suffered a stroke lead to improved neurite remodeling, neurogenesis and angiogenesis which in turn significantly improved the functional recovery of the subject [31]. A similar experiment in which intravenous administration of MSCs-generated exosomes enhanced angiogenesis and neurogenesis reduced the inflammation, improved spatial learning and sensory/motor function in a traumatic brain injury model [32].

### **2.6 Clinical applications**

At present, the use of autologous biologic injectates in the treatment of most acute and chronic conditions resulting in pain is considered investigational. There are currently 977 regenerative medicine trials worldwide, including gene therapy, cell therapy, and tissue engineering [2]. Of those, there at 51 and 66 clinical trials in the categories of musculoskeletal system and central nervous system, respectively. While a myriad of sources has reported positive results following the use of autologous biologic injectate, these reports are, overall, too heterogenous and underpowered to change the clinical practice of most. The absence of well-powered, level-1 data is demonstrating the efficacy of autologous biologic injectates may simply reflect the infancy of this field. Conditions recently studied for the use of autologous blood injectates include Complex Regional Pain syndrome (formerly called Reflex Sympathetic Dystrophy or RSD), OA, joint arthropathy including facet arthropathy and sacroiliitis, tendinopathies, degenerative disc disease, Multiple Sclerosis, headaches, migraines, and peripheral neuropathy. In this chapter we will discuss autologous biologic injectates in the treatment of OA, spondylosis, and tendon and ligament injury.

#### **2.7 Osteoarthritis**

Osteoarthritis (OA) is the most common joint disorder in the United States, and is estimated to 25% of people over 18-years old [33, 34]. OA is a progressive

**25**

way [44].

**2.8 Spondylosis**

*Regenerative Medicine*

*DOI: http://dx.doi.org/10.5772/intechopen.93717*

differences described were clinically relevant [43].

A new 5 year study published in 2019 demonstrates better outcomes and lower pain scores in patients who underwent knee arthroscopy for osteochondral knee injuries with and without preoperative intra-articular PRP injections. This research was the first study which used clinical data more than 5 years and demonstrates that cell therapy can promote the regeneration of articular cartilage in a lasting

Cervical and lumbar spondylosis represent the constellation of degenerative changes found in the cervical and lumbar spine that progressively occur in most people with aging. 25% of people under 40 years of age, 50% of people over 40 years of age, and 85% of people over 60 years of age are estimated to have cervical spondylosis [45]. Pathologically, the same degenerative processes that characterize OA extend to the vertebral joints and result in spondylosis often characterized by disc degeneration, uncinate spurring and facet arthrosis, ligamentous thickening and infolding, and deformity. Radiculopathy, myelopathy, discogenic pain, facet pain are the clinical manifestations of vertebral joint degeneration. Regenerative techniques for the treatment of cervical and lumbar spondylosis has been previously investigated with promising results. Further studies are needed to weigh safety profiles against efficacy. In an RCT of PRP vs. contrast for discogenic pain, Tuakli-Wosornu et al., reported significant improvements in pain and function at

disease affecting the joints and is characterized by perturbed immune responses to cellular injury resulting in cartilage degeneration, synovitis, bony remodeling, and chronic pain. Current treatment includes NSAIDs, opiate medications, topical analgesics, physical therapy, lifestyle modification, intraarticular steroid injections, intra-articular hyaluronic acid (HA) injection, and surgery. Non-steroidal antiinflammatory drugs (NSAIDs), opiates, and intra-articular steroid injections are primarily limited negative side-effects that accompany escalations in medication dose and frequency. Exogenous HA has been used as a treatment modality given observed decreases in endogenous HA exhibited in OA joints. However, due to a lack of consistent evidence, HA is not recommended by the American Academy of Orthopedic Surgeons in treatment of patients with symptomatic OA of the knee [35]. At present, the therapeutic value of PRP in treating OA is a topic of debate and investigation. Available studies suggest its clinical benefit compared with HA in treating OA of the knee [36–40]. Several authors have reported meta-analyses demonstrating improved pain and function following intra-articular injection of PRP in the knee versus HA [36–38]. In a meta-analysis of 26 randomized controlled trials involving 2430 patients, Tan et al. found better Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) total scores, WOMAC physical function, and Visual Acuity (VAS) Scores at 3, 6, and 12 months following PRP injection compared to HA [38]. Evidence supporting the use of PRP in hip OA is still lacking [39]. There is still much work to be done in understanding the therapeutic role of PRP in treating OA. It is an infrequently used treatment modality, a likely reflection of limited reimbursement. Presently, the Centers for Medicare and Medicaid Services (CMS) only cover PRP use when used for the treatment of chronic non-healing diabetic, pressure, or venous wounds in patients enrolled in clinical studies [41]. Despite growing public interest, available evidence does not support use of autologous stem cells in treatment of OA [42, 43]. In a meta-analysis of nine studies, evaluating 339 patients Huang et al. found most outcome measures similar between stem cell recipients and controls [43]. VAS was found to be statistically improved among stem cell patients; but, it is unclear whether the modest

#### *Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.93717*

*Pain Management - Practices, Novel Therapies and Bioactives*

sensory/motor function in a traumatic brain injury model [32].

ment of OA, spondylosis, and tendon and ligament injury.

At present, the use of autologous biologic injectates in the treatment of most acute and chronic conditions resulting in pain is considered investigational. There are currently 977 regenerative medicine trials worldwide, including gene therapy, cell therapy, and tissue engineering [2]. Of those, there at 51 and 66 clinical trials in the categories of musculoskeletal system and central nervous system, respectively. While a myriad of sources has reported positive results following the use of autologous biologic injectate, these reports are, overall, too heterogenous and underpowered to change the clinical practice of most. The absence of well-powered, level-1 data is demonstrating the efficacy of autologous biologic injectates may simply reflect the infancy of this field. Conditions recently studied for the use of autologous blood injectates include Complex Regional Pain syndrome (formerly called Reflex Sympathetic Dystrophy or RSD), OA, joint arthropathy including facet arthropathy and sacroiliitis, tendinopathies, degenerative disc disease, Multiple Sclerosis, headaches, migraines, and peripheral neuropathy. In this chapter we will discuss autologous biologic injectates in the treat-

Osteoarthritis (OA) is the most common joint disorder in the United States, and is estimated to 25% of people over 18-years old [33, 34]. OA is a progressive

**2.6 Clinical applications**

adipocytes [26]. MSCs were originally isolated from bone marrow, they have been isolated from other adult tissues such as adipose tissue, dental pulp, placenta, amniotic fluid, umbilical cord blood and Wharton's jelly, and even in the brain [27]. The MSCs differentiation potential for tissue repair has been studied extensively but the pattern of MSC mediated regeneration is now shifting toward secretomebased paracrine activity. The matter by which miRNAs play an essential role in physiological and pathological conditions is by regulating gene expression at the post-transcription level [28]. The pre-miRNA goes through an extensive biological process prior to maturation but an exosome can contain miRNA of different maturation stages and their release is a controlled process dependent on the source and developmental stage of derived cells rather than a random process [12]. It has been suggested that exosomes released by MSCs contain miRNA that control the microenvironment in the resident niches through a balance between proliferation and differentiation [29]. Additionally, tissue-specific responses have been described for exosomes isolated from different sources. For example, adipose tissue-derived exosomes seem to be more effective in halting the central nervous system degeneration caused by Alzheimer's disease when compared to bone marrow derived MSCs-derived exosomes [30]. But neurite outgrowth seems to be more responsive to exosomes released by menstrual fluid derived MSCs when compared to umbilical cord, chorion and bone marrow. Evidence of neurite outgrowth has also been shown in a middle cerebral artery occlusion model. MSCs exposed to ischemic cerebral extracts secreted exosomes containing mi-RNA that were transferred to neurons and astrocytes via exosomes and promoted neurite outgrowth and functional recovery. The same authors reported the use of cell free MSC-generated exosomes administered intravenously in a subject that had suffered a stroke lead to improved neurite remodeling, neurogenesis and angiogenesis which in turn significantly improved the functional recovery of the subject [31]. A similar experiment in which intravenous administration of MSCs-generated exosomes enhanced angiogenesis and neurogenesis reduced the inflammation, improved spatial learning and

**24**

**2.7 Osteoarthritis**

disease affecting the joints and is characterized by perturbed immune responses to cellular injury resulting in cartilage degeneration, synovitis, bony remodeling, and chronic pain. Current treatment includes NSAIDs, opiate medications, topical analgesics, physical therapy, lifestyle modification, intraarticular steroid injections, intra-articular hyaluronic acid (HA) injection, and surgery. Non-steroidal antiinflammatory drugs (NSAIDs), opiates, and intra-articular steroid injections are primarily limited negative side-effects that accompany escalations in medication dose and frequency. Exogenous HA has been used as a treatment modality given observed decreases in endogenous HA exhibited in OA joints. However, due to a lack of consistent evidence, HA is not recommended by the American Academy of Orthopedic Surgeons in treatment of patients with symptomatic OA of the knee [35]. At present, the therapeutic value of PRP in treating OA is a topic of debate and investigation. Available studies suggest its clinical benefit compared with HA in treating OA of the knee [36–40]. Several authors have reported meta-analyses demonstrating improved pain and function following intra-articular injection of PRP in the knee versus HA [36–38]. In a meta-analysis of 26 randomized controlled trials involving 2430 patients, Tan et al. found better Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) total scores, WOMAC physical function, and Visual Acuity (VAS) Scores at 3, 6, and 12 months following PRP injection compared to HA [38]. Evidence supporting the use of PRP in hip OA is still lacking [39]. There is still much work to be done in understanding the therapeutic role of PRP in treating OA. It is an infrequently used treatment modality, a likely reflection of limited reimbursement. Presently, the Centers for Medicare and Medicaid Services (CMS) only cover PRP use when used for the treatment of chronic non-healing diabetic, pressure, or venous wounds in patients enrolled in clinical studies [41]. Despite growing public interest, available evidence does not support use of autologous stem cells in treatment of OA [42, 43]. In a meta-analysis of nine studies, evaluating 339 patients Huang et al. found most outcome measures similar between stem cell recipients and controls [43]. VAS was found to be statistically improved among stem cell patients; but, it is unclear whether the modest differences described were clinically relevant [43].

A new 5 year study published in 2019 demonstrates better outcomes and lower pain scores in patients who underwent knee arthroscopy for osteochondral knee injuries with and without preoperative intra-articular PRP injections. This research was the first study which used clinical data more than 5 years and demonstrates that cell therapy can promote the regeneration of articular cartilage in a lasting way [44].
