Cadaveric tissue derived NABs

Bone, ligament, tendon and skin frequently are harvested from cadavers, processed, packaged and sterilized prior to distribution, which is beyond the scope of this review. However, the nucleus pulposus (NP) has been harvested from cadavers, from which NP progenitor cells have been isolated, and expanded in tissue culture to create a treatment for discogenic low back pain [8]. Treatment consists of a lyophilized NP allograft that is rehydrated just prior to injection, which is combined with a cryopreserved aliquot of cultured NP progenitor cells and injected into the disc through a 22-g needle. The presence of viable allogeneic cells in the treatment means it is a Section 351 category HCT/P and requires an IND/IDE and PMA to be completed prior to marketing in the USA. In a Level 1 study (Clinicaltrials.gov Identifier: NCT03709901), the cohort receiving the NP/Cells injection showed a durable decrease in both VAS (visual analog scale) and Oswestry disability index (ODI) at the 12-month milestone [8]. A post-hoc analysis of the data showed that study participants < 42 years old (the median age of the study participants) had a greater decrease in ODI compared to the saline cohort, as well as a higher proportion of responder-level participants (i.e., ≥ 15 points for ODI) compared to the saline cohort. In contrast, the older cohort didn’t show a similar pattern of response with the ODI metric. The incidence of adverse events (AEs) was similar in the two cohorts with rates of 30.9% for the < 42 year-old cohort and 28.8% for the ≥ 42 year-old cohort. The most common AE was back pain associated with either the procedure or treatment [9]. Details of these two studies are provided in Table 3.

Placental tissue derived NABs

As mentioned previously, placental tissue is a major source of NAB products used in treating a variety of pathologies, including wounds, tendons, nerve and bone, and are rich in extracellular matrix, cytokines, growth factors, proteoglycans and proteins [10]. Placental tissue derived products typically are made in various combinations of the amnion, chorion, and placental disk, and are available as fresh (hAM, hCM) or dehydrated (dhAM, dhACM) NABs [11, 12]. Commercially available human tissue derived NABs used for wound and burn care are shown in Table 4.

A recently introduced NAB for use in wound care is composed of the amnion and chorion, but includes the spongy layer that separates these two membranes in situ [dehydrated human amnion chorion membrane/spongy layer (dhACM/SL)] [13]. The spongy layer-containing sheet-form NAB was evaluated in a study of chronic non-healing wounds with various etiologies. The median time to heal was 77 days. Overall, treated wounds had the following levels of healing: 66% of the wounds had 100% healing, 5.7% had 70–99%, 9.4% had 40–70% and 18.9% had poor or no response. Venous leg ulcers, surgical wounds, and traumatic wounds had the highest response to treatment, while ischemic ulcer and pressure injury showed a poorer response. One factor that favored better response was observed for wounds with less than 1 year chronicity [13]. Additional details for this clinical study are provided in Table 3. In a Level 1, multi-clinic study of a dehydrated human amnion chorion membrane (dhACM) product for treating diabetic foot ulcers that were refractory to standard of care, it was found that 70% of the study participants receiving the dhACM product had complete wound closure at 12 weeks compared to just 50% of the participants in the control cohort. The rate of complete closure increased to 95% at the study endpoint of 16 weeks for the dhACM cohort, while just 86% of the control cohort had complete closure [14]. Additional details for this clinical study are provided in Table 3. One area where wound closure is a serious challenge is in treating pediatric burns. A commercially available dhACM graft was used to treat 30 patients with a variety of superficial partial to full thickness burns in place of using a split thickness skin graft (STSG). Healing was enhanced and reported to be faster compared to published reports using STSGs, while pain also was reduced [15]. Additional details for this clinical study are provided in Table 3. Several commercially available dhACM products were compared in a review of published clinical studies in terms of clinical outcomes and use [12]. The sheet form of placental tissue derived NABs also has been used to treat dermatologic conditions that can become chronic wounds. In a review of published case series involving dermatology conditions, dhACM was reported to reduce healing time and pain, and resulted in better cosmetic outcomes [16].

In addition to the sheet form of placental tissue derived NABs discussed above, there are a number of NAB products that are made by micronization of placental tissue, which renders them suitable for injection; no injectable NAB products outside of a single product for use in wound care have been approved by the USFDA so far. Micronized amniotic membrane (AM)-only NAB products are referred to as “amniotic suspension allograft” (ASA) [17], while micronized dehydrated amnion/chorion membrane products are labeled as micronized dehydrated human amnion chorion membrane (mdhACM) [18]. A Level 1 randomized control trial (Clinicaltrials.gov Identifier: NCT01659827) was performed to assess safety and efficacy of a mdhACM product to treat chronic plantar fasciitis [18]. Two doses of the mdhACM were injected, with saline as a control. At the study endpoint of 8 weeks, the control cohort had an average improvement of 12.9 points on the American Orthopedic Foot & Ankle Society Hindfoot metric, while both mdhACM treated cohorts reported an improvement greater than 50 points. No AEs related to the injections were reported [18]. Additional details for this clinical study are provided in Table 3. The use of an injectable, cryopreserved, AM NAB product [cryopreserved human amniotic membrane (c-hAM)] was evaluated in a Level 1 randomized control study for treating plantar fasciitis, with corticosteroid injection as the control. At the 12-week endpoint, there was no meaningful improvement of the c-hAM cohort over the control. However, a small group of participants in each cohort opted for a second injection for which the primary outcome metric of the Foot Health Status Questionnaire was meaningfully improved at the 18-week milestone for the 2-dose c-hAM treated cohort. No AEs associated with the treatments were reported [19]. Additional details for this clinical study are provided in Table 3. A mdhACM product was evaluated for safety and efficacy in treating knee OA in a retrospective case series study of 82 consecutive patients. The primary outcome measure was the Knee Injury and Osteoarthritis Outcome Score (KOOS), whose subscales (QoL and pain) showed a range of improvement from 55% to 118% over baseline at the 6-month endpoint. AEs were reported by 68% of participants, which resolved within 2–7 days post-injection [20]. Additional details for this clinical study are provided in Table 3. An ASA product was evaluated in a randomized clinical study for treating knee OA (Clinicaltrials.gov Identifier: NCT02318511), with saline and hyaluronic acid (HA) as comparators [17]. There were 68 patients treated with ASA, 64 with HA and 68 with saline. The QoL subscore of KOOS was meaningfully improved for the ASA cohort over the HA and saline cohorts out to the 12-month endpoint, but there was no consistent pattern of improvement for the other KOOS subscores. All VAS subscores were meaningfully improved for the ASA cohort over the HA/saline cohorts at the 12-month endpoint. Overall, based on the Outcome Measures in Rheumatology and Osteoarthritis Research Society International (OMERACT-OARSI) criteria for assessing responders, there were 50%, 25% and 25% high responders for the ASA, HA and saline cohorts, respectively. Treatment-emergent AEs were reported by 2.9% and 3.0% of the ASA cohort and the HA cohort, respectively [17]. Additional details for this clinical study are provided in Table 3. A pilot study of the safety and efficacy of treating knee OA (n = 25) with a non-commercial ASA was performed [21]. The in-house ASA agent was prepared by a medical institute with donor screening and microbial sterility testing of the ASA product. No SAEs were reported, although 16% of the study participants reported mild AEs, which resolved within a few days. The International Knee Documentation Committee (IKDC) score and VAS both showed a plateauing of improved scores from the 6- to 12-month endpoints, with both showing a meaningful improvement compared to baseline at the 12-month endpoint [21]. Additional details for this clinical study are provided in Table 3. Finally, a commercially available ASA was used to treat hip OA in a prospective study of ten participants with a 1-year endpoint [22]. One participant left the study at 2 months post-injection and received a total hip replacement. The remaining participants reported meaningful improvements compared to baseline for the modified Harris hip score (HHS; mHHS) and the International Hip Outcome Tool at the 12-month endpoint. Only the “maximal pain over the previous three days” VAS subscore was meaningfully improved compared to baseline at the 12-month endpoint. None of the patients remaining in the study reported any treatment related AEs [22]. Additional details for this clinical study are provided in Table 3.

Cultured cells as therapeutic treatments

Cells isolated from a variety of tissues, including bone marrow, adipose tissue (AT) and umbilical cord tissue, have been cultured to create allogeneic cell therapies to treat OA, low back pain and other painful and degenerative tissue pathologies. A recent review of randomized control trials highlighted the variations in using cultured allogeneic mesenchymal stromal cells (MSCs) to treat knee OA [23]. The allogeneic cell trials used cultured MSCs obtained from AT, bone marrow, placental membranes and Wharton’s Jelly. The cell dose injected as well as the number of injections also varied. Despite the variables of dose and OA conditions, five of the six studies reported favorable improvements in pain and disability metrics. In one study, one of the treatment arms involved injecting an allogeneic MSC agent at monthly intervals to treat knee OA, but the study was stopped due to increasing pain at the site of injection [23]. The use of bone marrow derived cultured MSCs to treat discogenic low back pain has been evaluated in two clinical studies. An allogenic dose of 25 × 10⁶ MSCs was injected intradiscally in the cell treatment cohort, while the control group received a sham injection of anesthetic in the paravertebral muscles [24]. At 42-month follow-up, the MSC-treated cohort had VAS and ODI scores that were meaningfully improved compared to baseline, while the control cohort VAS and ODI scores weren’t improved compared to baseline. Importantly, the Pfirrmann grades of the treated discs were assessed periodically (by MRI), which revealed a reduction in the Pfirrmann grade of 0.6 for the MSC-treated cohort, whereas there was an increase in the Pfirrmann grade of 1.0 for the control cohort [24]. Additional details for this clinical study are provided in Table 3. A bone marrow derived allogeneic MSC therapy in the process of commercialization was evaluated in a Phase 1b/2a study (Clinicaltrials.gov Identifier: NCT01290367) to assess safety and efficacy of treating discogenic low back pain with 6 × 10⁶ MSCs suspended in a 1% HA solution compared to saline as a control [25]. At the 36-month milestone, 43% of the participants treated with MSCs and 20% of saline-treated participants showed a 50% decrease in VAS. A total of 20% of the MSC-treated cohort reported AEs, with none being characterized as a SAE. In contrast to the other allogeneic MSC intradiscal study reviewed above, no changes were reported in the modified Pfirrmann grades of any of the participants receiving the allogeneic MSC treatment [25]. Additional details for this clinical study are provided in Table 3.

Not all allogeneic therapeutic cell preparations are obtained from adipose, bone marrow or placental tissues: NP cells were recovered from live donor intervertebral discs and cultured to create a discogenic cell-based therapy. The cultured discogenic cells were evaluated for safety and efficacy in a Phase 1/2 IND randomized control trial (Clinicaltrials.gov Identifier: NCT03347708) for treating discogenic low back pain [26]. A low dose (3 × 10⁶ discogenic cells), a high dose (9 × 10⁶ discogenic cells), hyaluronate (vehicle) and saline (placebo) were used to treat 20, 20, 10 and 10 study participants, respectively. The high dose cohort showed a meaningful difference in VAS and ODI outcomes compared to baseline starting at 1-year and continuing through the 2-year endpoint. The high dose cohort also had the highest number of participants (70%) with a > 30% reduction in VAS compared to 60% for the combined hyaluronic/saline cohorts and 55% for the low dose cohort. The high dose cohort showed a meaningful decrease in ODI that exceeded the minimal clinically important difference (MCID) of –15 points at 12-, 26-, 78- and 104-weeks, while the other three treatment arms failed to reach the MCID. Changes in disc volume assessed by MRI showed a mean increase in disc volume of approximately 400 mm³ for the high dose cohort, while the other three cohorts showed a loss in disc volume (hyaluronic, saline), or no net change in volume (low dose) at the 104-week milestone. The frequency of AEs was lowest for the high dose cohort. There was an incidence of 6.7% for SAEs, which were associated only with study participants in the saline and hyaluronate groups. Overall, the use of an allogeneic cell agent derived from the NP demonstrated efficacy in reducing pain and improving QoL, as well as achieving a durable increase in disc volume out to the 2-year endpoint [26]. Additional details for this clinical study are provided in Table 3.

Cell culture derived products

Conditioned medium recovered from the culturing of a variety of progenitor cells (e.g., MSCs) has been evaluated as a source of therapeutic treatments. Cultured cells release biomolecules and EVs into the tissue culture fluid, which is referred to as the cell’s “secretome” [7, 27, 28]. The excitement surrounding the use of conditioned media as a therapeutic agent is based on the realization that most of the therapeutic benefit attributed to MSCs is associated with the wide variety of biomolecules secreted by the MSCs in vivo, which is referred to as the paracrine effect [29]. Thus, a secretome containing NAB product might offer an opportunity to provide an allogeneic cell-free regenerative therapy, which reduces the risk of immune rejection of the allogeneic cells [27]. However, as is the case with any cultured therapeutic product intended for injection, there is a requirement to produce the secretome under CGMP (current good manufacturing practice) conditions to ensure sterility and safety. The effort to produce a secretome in a lyophilized form under CGMP conditions at a pilot scale has been reviewed [30]. One emerging issue relates to the differences among the secretomes obtained with MSCs isolated from various tissues. In vitro assessments of the secretomes of cells isolated from AT and AM were found to have differential impacts on cultured cell-based models. For example, AT-conditioned medium was shown to induce higher proliferation and better supported neurite outgrowth, while AM-conditioned medium displayed a profile of greater immunomodulatory activity, migration and re-growth of cells in a scratch model assay [31]. In another report, secretomes obtained from umbilical cord MSCs, bone marrow MSCs and AT MSCs were found to have variations in the presence of important biomolecules, as well as in the amount of these molecules in the secretomes studied [28]. While almost all of the therapeutic evaluations of the potential clinical benefits of secretomes are limited to in vitro and pre-clinical studies, there was a recent report on the use of a CGMP-produced secretome to treat severe COVID-19 patients [32]. The Level 1 trial was double blinded, and placebo controlled. Treatment consisted of a single IV infusion of 15 mL of the secretome product in 100 mL of saline, while the control group received the same volume of saline. One of the few clearcut differences reported in the study occurred in the change in the ratio of the level of IL-6 (interleukin-6, pro-inflammatory) to IL-10 (anti-inflammatory) on D14 post-treatment. Control patients showed a meaningful increase in the ratio from D7 to D14, while the ratio of these cytokines for the intervention patients didn’t increase [32]. Additional details for this clinical study are provided in Table 3. An important consideration to keep in mind when thinking about using secretome products is their efficacy compared to the efficacy of the source cultured cell, which is the focus of a very recent study [27]. The secretome dermal stem/stromal cells (seDSCs; adherent cells isolated from human skin) and secretome AT stem/stromal cells [secretome adipose stem/stromal cells (seASCs); adherent AT derived cells isolated from the skin donor] were analyzed and used in a surgical wound murine model. The secretomes were found to share 663 proteins, while 102 proteins each were unique to the dermal stem/stromal cell (DSC) and adipose stem/stromal cell (ASC) derived secretomes. In a pre-clinical study of skin wound healing in a murine model, the secretomes from the ASCs and DSCs were compared with the cultured cells that produced the secretomes, along with a negative control of a commercially available collagen-based scaffold that was used as a scaffold for the treatments. The degree of wound closure on D21 for the treatments was as follows: DSCs (90.3%) > ASCs (86.9%) > seASC (68.1%) ≈ seDSC (67.3%) > Scaffold (55.8%). Wound closure with the DSC and ASC treatments were meaningfully improved over the scaffold, while the seASC and seDSC treatments were not. As noted in the publication, this is the first paper in which both the parent cell and its secretome were assessed for efficacy in a pre-clinical model [27].

Blood and plasma derived products

Autologous platelet-rich plasma (PRP) is a frequently prescribed therapy for a broad spectrum of MSK pathologies. Since it is an autologous therapy, the patient’s condition might limit the potential therapeutic benefit, which is of concern with diabetics, smokers, the obese, the elderly and those patients with a low platelet count [33]. As an extension of the availability of platelet concentrates (PCs) in blood banks for use in hemostatic support, expired PC units have been used to create platelet lysates (PLs) and are a source of exosomes [34]. There are a few reports that describe the use of allogeneic PRP in treating MSK pathologies. The use of allogeneic PRP to treat knee OA in patients suffering from primary immune thrombocytopenia was evaluated in a randomized control trial [35]. Eighty study participants were randomized into an allogeneic PRP (allo-PRP) treatment cohort or a saline placebo cohort. The allo-PRP was obtained from a single healthy donor. One injection of either the allo-PRP or saline was delivered via the intraarticular route. No hemoanalysis of the allo-PRP injectates was performed, although the device used was cited as producing a leukocyte-poor allo-PRP. The primary outcome metric was Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Over the course of the 1-year study period, the allo-PRP cohort reported meaningfully improved WOMAC results only at the 3-month milestone compared to the saline cohort. The frequency of short-term AEs in the allo-PRP cohort was 10% and in the saline cohort was 7.5%, which resolved without further intervention within a few days. However, more study participants reported durable pain and swelling in the injected knee through the 12-month endpoint of the study with the allo-PRP treatment compared to the saline treatment. There were no SAEs reported in the study [35]. Additional details for this clinical study are provided in Table 3. The use of cord blood as the source of platelets has been explored to treat hip OA via an intraarticular delivery as reported in a retrospective case review [33]. Patients were treated with either an injection of autologous PRP (A-PRP) or blood-typed/matched allogeneic cord blood derived PRP (C-PRP) once per week for three weeks. Cord blood was obtained from donors with uncomplicated deliveries or C-sections. Homologous cord blood units were pooled and subsequently processed by a manual method to yield three aliquots of C-PRP for use in treating a participant, which was stored frozen. A similar process was used with the autologous blood processing to yield three aliquots of A-PRP, which also were stored frozen. In addition to the use of blood-typed matched C-PRP, the cord blood was processed through a leukocyte-depletion filter, while the A-PRP wasn’t, in order to reduce the risk of immune rejection. The frequency of AEs was not meaningfully different between the two PRP treatment cohorts, and these AEs were of short duration following the injection. There were no meaningfully improved differences in outcomes at any milestone between each type of PRP and baseline. However, if data was stratified according to the patient’s extent of hip OA score (Tonnis metric 1–2 vs. 3), C-PRP patients with low grade hip OA had a meaningful improvement for the HHS at the 12-month endpoint compared to the HHS score for the A-PRP cohort [33]. Additional details for this clinical study are provided in Table 3.

Young plasma

The use of young plasma by humans as a means of combating age-related decline (“anti-aging”) is based on results obtained in a parabiosis rodent model, in which the circulatory systems of young and old mice were surgically connected. Results from a number of parabiosis experiments over the past two decades have demonstrated the benefit of “young plasma factors” on the older mice’s pro-inflammatory status [36], a condition referred to as “inflammaging” [6]. Analysis of the protein contents of “young plasma” have led to the identification of key biomolecules that are thought to play a role in modulating the negative aspects of getting old. For example, apelin is a hormone that is characterized as a “rejuvenating factor”, since it is at higher levels in younger mice and decreases as mice age. β2-Microglobulin is a “pro-aging” factor, which is found at higher levels in older mice, so its reduction would contribute to slower aging [36]. The devil, of course, is in the details in view of the complex feedback loops and interacting metabolic pathways that manage a person’s physiology as they age. Based on the evidence emerging from pre-clinical model studies, it seems like young plasma could be considered as the biological equivalent of a compounding pharmacy. Unfortunately, so far there are virtually no publications on human clinical studies performed at any level to support the potentially beneficial anti-aging effects of young plasma. However, in a very recent publication [6], the influence of a 5% plasma protein fraction derived from young donors (the average age of donors was 35 years old) that is in the process of being commercialized was evaluated in patients undergoing a planned surgery. The Phase 2a IND trial (Clinicaltrials.gov Identifier: NCT 03981419) enrolled patients that would be undergoing knee or hip arthroplasty in order to assess the influence of the 5% plasma agent on the patients’ response to surgical injury. Patients were randomized to receive either plasma or saline infusion, with both cohorts receiving a series of infusions starting the day before surgery, before and after the surgery itself and the day after surgery. Proteomic and cell analyses were performed to assess patterns of up- and down-regulated proteins and types and numbers of cells. Both cellular and protein profiles showed changes that supported anti-inflammatory immune modulation, along with beneficial changes in other supporting pathways. In addition, patients were surveyed for fatigue, impairment of daily function and pain following their surgery. While none of the outcomes were meaningfully different between the two cohorts, the median time to reach “mild” pain in the plasma protein fraction-treated cohort was 12 days, while the same milestone took 18 days for the saline cohort [6]. Additional details for this clinical study are provided in Table 3.

EVs (exosomes)

EVs are released by cells in the body in three ranges of diameter: 30–150 nm (exosomes); 150–500 nm (microvesicles); and 500–800 nm (apoptotic bodies). Exosomes contain “cargo” that has been shown to mimic the paracrine effects of the parental MSCs that produce the exosomes. Consequently, MSC derived exosomes have dominated efforts for commercialization, with almost all exosome preparations being derived from bone marrow, AT or umbilical cord tissue [37]. Furthermore, all of the fourteen active clinical studies recently listed on Clinicaltrials.gov that used exosomes as the therapeutic agent were obtained from those same three tissues [37]. The roles played by specific components of exosome cargo, like microRNAs and proteins, in MSK health and regulation have been reviewed [38]. For example, several microRNAs have been found in exosomes released by mineralizing osteoblasts, which were shown to promote differentiation of a murine cell type (ST2) into an osteoblastic lineage [38]. While cultured MSCs release exosomes with cargoes that can act to reduce inflammation, promote resident tissue cell proliferation and enhance angiogenesis (among other effects), a very active area of investigation involves functional modification of exosome cargoes through manipulation of the culture conditions (e.g., hypoxia, additives in the culture medium, etc.) [39]. Improved delivery of exosomes also is being explored based on modifying the surfaces of exosomes to enhance binding to or interacting with hydrogels or other types of carriers [39]. Although the clinical trial doesn’t involve an MSK pathology, clinical outcomes of a significant Phase 2 IND trial (Clinicaltrials.gov Identifier: NCT04493242) of a bone marrow MSC derived exosome-based therapeutic agent recently were published on treating acute respiratory distress syndrome (ARDS) in hospitalized COVID-19 patients [40]. Patients in the active treatment cohort were treated with two doses of the exosome product containing 1.2 × 10¹² exosomes/dose, which was diluted in approximately 100 mL of saline and delivered via IV, while the placebo cohort received 100 mL of saline. The exosome product-treated cohort had a shorter time to discharge, and ventilation-free days were meaningfully higher compared to the placebo cohort. As shown in Table 5, while the All-cause mortality in the exosome product-treated cohort was not meaningfully improved compared to placebo for All COVID-related ARDS patients, a post-hoc analysis of the mortality rates for the moderate to severe ARDS patients showed a meaningfully improved outcome of 30.8% for the exosome product-treated cohort compared to 72.7% for the placebo cohort [40]. Additional details for this clinical study are provided in Table 3. Based on the reduction in mortality shown in the Phase 2 IND, the USFDA agreed to allow the Phase 3 trial to start in 2022, and in 2023 agreed to extend the scope of the Phase 3 trial from COVID-19 related ARDS to ARDS of any etiology [41]. The bone marrow MSC derived exosome product under investigation is the first exosome-based therapeutic agent to progress to a Phase 3 IND trial in the USA.

Composite cell culture-based constructs

There are two examples of cells being cultured on carriers to create a composite cell-carrier wound care treatment that are USFDA approved. Stratagraft^® (Stratagraft Corporation, Madison, WI, USA) is comprised of an allogeneic cellularized scaffold (rat collagen), which is conditioned by human dermal fibroblasts and seeded with a human-derived keratinocyte cell line. Stratagraft^® is placed over the debrided burn, which allows the patient’s cells to migrate into and remodel the construct. In a summary of the Phase 3 IND trial (Clinicaltrials.gov Identifier: NCT03005106), Stratagraft^® was able to accelerate wound closure in 83% of the study participants by month 3, without the use of an autograft supplemental treatment, which eliminates the risk for donor-site morbidity and wound healing complications in patients with deep partial-thickness burns [42]. Additional details for this clinical study are provided in Table 3. Apligraf^® (Organogenesis Inc, Canton, MA, USA) is a composite wound care product that is comprised of a bilayer having human keratinocytes growing as a well-defined epidermal layer, and human fibroblasts in a bovine collagen scaffold on the dermal side [43]. A review of clinical studies in which Apligraf^® has been evaluated for treating diabetic foot ulcers and other partial- or full-thickness skin ulcers generally showed slower healing rates compared with other NABs [12].