A Comprehensive Guide of Cellular Blood-Derived and Mesenchymal Stem Cell-Based Autologous Biological Preparations for Tissue Repair, Regeneration, and Wound Healing

Abstract

The use of autologous biological preparations (ABPs) and their combinations fills a void in health care treatment options that exists between surgical procedures and current pharmaceutical treatments. There is a wide range of ABPs that can safely and effectively be prepared at point of care using tissues from the patient such as peripheral blood, bone marrow, and adipose tissue to treat a wide range of clinical conditions. The use of blood-derived and mesenchymal stem cell cellular preparations plays important roles in the modulation of tissue repair processes in complex biological settings. Biological products derived from autologous tissues are advantageous because of their autologous nature and their safety profiles. ABPs include platelet-rich plasma (PRP), bone marrow concentrates (BMCs), and adipose tissue complex (ATC) with its unique stromal vascular fractions (SVFs). In addition, ABPs can be combined to create biological preparations that are more diverse and possess a high degree of regenerative activity and potential. Likewise, concentrated acellular plasma proteins can generate a temporary fibrin matrix to interact with the bioactive molecules of various ABPs. Practitioners reason that the application of ABPs can mimic the classical healing and angiogenesis cascades to initiate tissue repair, regeneration, and wound healing during non-surgical interventions, aiming to restore the integrity and function of damaged tissues.

1. Introduction

Various pathological conditions, chronic recalcitrant wounds, damaged tissue structures, or degenerative tissues may cause wound healing and tissue repair to be highly complex and dynamic processes. These conditions dependent upon local and systemic multicellular biological processes including cell signaling [1, 2, 3, 4]. The application of autologous biological preparations, prepared at point-of-care, hold the promise of promoting tissue healing, regeneration, and repair in a natural fashion [5]. Blood-derived and mesenchymal stem cell-based preparations play key roles in tissue repair mechanisms and the modulation of wound healing processes in complex biological microenvironments. Moreover, plasma and concentrated proteins in a provisional fibrin matrix can interact with bioactive molecules and cells and controlling the spatial-temporal distribution of these molecules and cells [6]. There is an unmet need in treatment options of degenerative and traumatized tissues, difficult-to-heal acute and chronic wounds. Regenerative medicine is an area of specialized, interventional, and non-surgical medicine, often not achievable via surgery of medications. In general, ABPs for therapeutic interventions are increasingly being utilized to support the healing of chronic wounds and the repair and regeneration of tissues by delivering allogeneic or autologously prepared biological cells.

The purpose of this chapter is to provide a comprehensive overview of the most widely used cellular autologous blood-derived, bone marrow-derived, and adipose-derived mesenchymal stem cell-based biological therapies, as well as some combinations of these therapies in tissue repair, regeneration, and wound healing treatment strategies.

2. Positioning ABPs

Traditionally, health care treatment options consist of a variety of surgical procedures and techniques and the use of medicinal treatment protocols and formulations. Alternatively, regenerative medicine therapies have been introduced, using biological treatment strategies to enhance healing and improve symptoms in many tissues in a large variety of medical conditions [7]. These non-surgical treatment options are referred to as interventional autologous biologically based procedures. In many instances, regenerative medicine procedures result in adequate patient outcomes that are often not achievable via surgeries or the use of medications, as shown in Figure 1.

Figure 1.

Schematic representation of some characteristics and options of current health care treatment options, including the use of ABPs for tissue repair, regeneration, and wound healing as non-surgical interventions therapies. Additionally, ABPs are also used in combination with surgical procedures (Biosurgery) to achieve improved patient outcomes.

ABPs can be safely derived from several freshly acquired biologically derived materials, originating from human blood, bone marrow, or adipose tissue, at point of care [8].

2.1 The biology of tissue repair, tissue regeneration, and wound healing using ABPs

ABPs are live cellular and stromal biomaterials that include a wide variety of platelet-rich plasma (PRP), bone marrow concentrate (BMC), and adipose tissue-derived tissue SVF (tSVF) bioformulations. The foremost advantages of autologous biological products (ABPs) are their autologous nature and their safety profile [9].

Within these specific ABPs, a diversity of bioformulations can be prepared to treat different tissue types and pathologies. An overview of the most cited cells, stroma, bioactive matrix and molecules that play significant roles in non-surgical biological interventional procedures to induce cell-cell communication, cellular and molecular signaling in a treated pathological microenvironment has been published extensively by us [10]. Once precisely delivered, biologics will locally initiate and regulate biological mechanisms and pathways, to interact with the local niche resident cells that potentially lead to tissue repair, tissue regeneration, and wound healing. For these reasons, these multi-cellular heterogeneous cell concentrates have become a valuable biological instrument to treat non-surgically complex pathologies. Furthermore, these preparations are often used to enhance the results of bio-surgical procedures [2, 11].

2.2 The role of ABPs in tissue healing

Autologous biological technologies are designed to mimic the initiation of the classical and angiogenesis healing cascades in a wide-ranging field of medical applications [12]. Ultimately, tissue repair, tissue regeneration, and wound healing are complex biological processes aimed at restoring the integrity and function of damaged tissues. These processes follow well-defined cascades of events, with a magnitude of systemic and local cellular activities taking place in a sequential manner.

The traditional healing cascade can be categorized into distinct phases, each characterized by specific cellular activities. The initial phase involves hemostasis, which is achieved by the aggregation of platelets at the site of tissue injury, promoting blood clotting while forming a fibrinous network. Thereafter, platelets play a crucial role in regulating the healing process by secreting their granular content and releasing a magnitude of platelet growth factors (PGFs), cytokines, and molecules capable of initiating tissue repair [9, 13]. The lesser-known angiogenesis cascade adds an additional layer of complexity to the wound healing process. It may not have been studied as extensively as the traditional wound healing cascade, but it plays an imperative role in wound healing because it involves recruitment of endothelial cells, differentiation, proliferation, and ultimately formation of new blood vessels from preexisting ones (angiogenesis) [14].

3. Outlining ABPs

Many biological treatments options are available to treat various clinical maladies (Figure 2). The preparation of ABPs can be categorized into cellular preparations (such as tSVF, cSVF, or culture expanded cell groups), acellular preparations, or combinations of these methods. Cell-based preparations can be classified into two categories: preparations derived from peripheral whole blood and preparations derived from stromal vascular fraction elements, including mesenchymal, pericytes, endothelial, peri- and paravascular stem cells, originating from bone marrow or adipose tissue. Preparations derived from blood are available in various compositions and characterizations, such as PRP, platelet-rich fibrin (PRF), platelet lysates (PL), or as a combined PRP and plasma protein preparation [15, 16].

Figure 2.

A simplified overview of ABPs which are available to treat an abundance of clinical pathologies. In this figure, only autologous prepared products, prepared at point-of-care, and a combination of these products are shown. MSC: mesenchymal stem cell; PRP: platelet-rich plasma; PRF: platelet-rich fibrin; PL: platelet lysate; BM: bone marrow; AT: adipose tissue; P-PRP: pure PRP; NP-PRP: neutrophil-poor PRP; NR-PRP: neutrophil-rich PRP; PRP-G: PRP Gel; i-PRF: injectable platelet-rich fibrin; A-PRF: activated platelet-rich fibrin; L-PRF: leukocyte platelet-rich plasma; PRGF: platelets rich in growth factors; PRFM: platelet-rich fibrin matrix; BMA: bone marrow aspirate; BMAC: bone marrow aspirate concentrate; ATC: adipose tissue complex; cATC: compressed ATC; t-SVF: tissue stromal vascular fraction; c-SVF: cellular stromal vascular fraction; ACS: autologous conditioned serum; A2M: alpha-2-marcoglobulin; PPP-M: platelet-poor plasma matrix; PR-PPP: protein-rich platelet-poor plasma; PR-PRP: protein-rich platelet concentrate.

Bone marrow aspirate (BMA) and BMC are frequently cited as autologous MSC and progenitor cell preparations [17, 18]. Adipose tissue-based preparations can be defined as autologous ATC, concentrated ATC, tissue stromal vascular fraction (t-SVF), and cellular stromal vascular fraction (c-SVF) [19, 20].

Acellular preparations consist of (concentrated) plasma proteins and protein fibers, containing a combination of insoluble and soluble molecules. Protein-based technologies are a class of natural biomaterials that can be used either as standalone products (fibrin glue and sealant) or as scaffolds in combination with cellular-based products [21, 22].

3.1 Blood-derived biological preparations

Blood-derived platelet preparations are prepared from a unit of whole blood by centrifugation methods to facilitate gravitational cellular density separation [10]. The final prepared biomaterials are collectively termed autologous platelet concentrates.

The employment of autologous platelet concentrates has been of interest to clinicians and researchers for decades because of their potential to accelerate and support in numerous cellular activities that can lead to tissue repair, tissue regeneration, wound healing, and ultimately functional structural repair. However, contingent on the PRP device used, platelet preparations can contain varying platelet concentrations, as well as differences in bioformulations. It has been suggested the low-quality platelet concentrates, incorporating low platelet numbers and inconsistent other cellular content, are directly responsible for poor patient outcomes [23, 24, 25, 26, 27].

The differences in composition between PRP, plasma-based PRP preparations such as PRF, and blood plasma were described in a proteomic analysis [28]. A total of nearly 600 proteins were detected in human plasma, and an in-depth analysis revealed that PRP formulations contained significantly more proteins than both platelet poor plasma-based PRP products and whole blood plasma.

Although more positive than negative patient outcomes have been reported in the literature, no definitive and accepted standards have been realized for preparing PRP preparation methods and product classifications. A simplified categorization would be to divide platelet concentrates in PRP-buffy coat and plasma-based PRP technologies. The PRP technologies exploit higher patient whole blood volumes to prepare the final PRP products with higher platelet concentrations and preparation options in bioformulation.

Depending on the PRP device architecture, the flexibility to produce different bioformulations, and the employed centrifugation protocols, PRP device uses one spin or two spin centrifugation cycles [29]. Following the PRP manufacturer’s instructions for use, and the practitioner’s desired PRP bioformulation, PRP devices should be able to sequester a unit of freshly anticoagulated whole blood into a clear PPP fraction, a buffy coat stratum, and a red blood cell fraction, as illustrated in Figure 3. Indicatively, these PRP-based systems are capable of capturing platelets after processing that will increase the PRP platelet concentration between five to more than 12 times the baseline whole blood platelet concentration [30].

Figure 3.

An image of PRP preparation after the second centrifugation step. The concentrated platelets are present on the top of the buffy-coat stratum (device in picture is the PurePRP®SP, EmCyte Corporation®, Fort Myers FL, USA). PRP: platelet-rich plasma; PPP: platelet-poor plasma.

Alternatively, plasma-based devices utilize low patient blood volumes to a produce a low volume “PRP-like” product. These systems use mostly a single spin processing protocol designed to mainly isolate the PPP fraction from the whole blood, incorporating platelets that are present in this fraction. Their final platelet concentration ranges from 0.5 to 2.5 times the native platelet concentration [31].

As with PRP, plasma-based products can further be classified into leukocyte- and platelet-rich fibrin (L-PRF) and leukocyte-poor or pure platelet-rich fibrin (P-PRF) [32].

3.1.1 Buffy-coat-based PRP technology is rich in platelets

A variety of PRP bioformulations, terminologies, and product descriptions have been introduced to practitioners over the years [15, 31, 33]. As part of the healing and angiogenesis cascades, platelet biological components and mechanisms are crucial to tissue repair, regeneration, and wound healing [14]. In response to administered PRP and the subsequent in situ tissue platelet activation, a number of regenerative and mitogenic components are released, which initiate cell differentiation, proliferation, and metabolic activity [34].

In the absence of consensus on standardization and classification of PRP devices and preparation methods to produce consistent effective PRP formulations, approximately 20 buffy coat PRP devices have been introduced to the market. These PRP devices can be categorized as pure PRP, leukocyte-poor PRP (LP-PRP), or leukocyte-rich PRP (LR-PRP) [35]. Considering the vast differences between the architecture of PRP devices, a great deal of variability has been reported on PRP platelet dosing and cellular characterization, which has led to poor and even negative patient outcomes [36].

As a best practice, different PRP formulations should be used at the same time in the same patient to treat multiple pathoanatomic conditions by using tissue-type-specific PRP formulations to optimize microenvironmental healing response and ultimately restore tissue structures that facilitate functional tissue repair [36], as portrayed in Figure 4. In particular, when treating chronic wounds, different PRP formulations should be utilized, based on the wound bed tissue appearances [5].

Figure 4.

A graphic illustration showing the administration of multiple different PRP formulations to treat various pathoanatomic tissue structures in the same patient, during the same session, to optimize total joint healing [36].

3.1.2 Plasma-based PRP technologies are poor in platelets

In plasma-based PRP procedures, a “PRP-like” suspension is prepared, excluding erythrocytes, and in most instances leukocytes [37]. In PRF plasma-based preparations, fibrin is the most important constituent of the final injectate [38]. This blood-derived plasma-based PRP product represents a plasma protein matrix which can contain processed platelets and eventual other cells that have cell stimulatory and immunological properties.

In more specific terms, this ABP consists of a fibrin-matrix compound containing platelets, with or without leukocytes, and other molecules [6, 39]. A notable aspect is that many plasma-based test-tube devices do not use anticoagulants when harvesting patient’s blood prior to processing. It is anticipated that platelet activation will occur, leading to the release of clotting factors, including thrombin that will convert the PPP fibrinogen into a fibrin matrix after processing [40, 41], shown in Figure 5. It has been demonstrated that fibrin polymerization, which is often induced by the addition of calcium chloride, results in a denser fibrin matrix when compared to non-activated PRP [42]. Thus, following therapeutic application, fibrinolysis of the cellular fibrin matrix will be initiated by plasminogen and plasmin, thereby liberating fibrin matrix components for a prolonged period of time compared to activated PRP clots which do not contain excessive amounts of plasma fibrin and other proteins [22].

Figure 5.

Plasma-based test tube preparation (A). After a single centrifugation procedure, the whole blood (10 ml) is segmented and clotted. The clot is extracted from the test tube, and the erythrocyte fraction is separated from the clot, leaving a fibrin matrix for clinical application (B).

In two major device comparison studies, Fadadu and Magalon evaluated many PRP devices, and they concluded that the platelet concentration was significantly lower in the low volume and single spin plasma-based PRP devices, when compared to buffy-coat PRP-based devices [31]. The results of these studies have been confirmed by others who have measured higher PGF concentrations following high preparation volume devices and double spin procedures [43]. Therefore, one might assume that the tissue repair, regeneration, and the wound healing capacities are less in plasma-based devices when compared to buffy-coat PRP systems. A striking observation is that some of the plasma-based platelet rich fibrin (PRF) devices have been presented with more than 20 different sub-formulations and preparation protocols by modifying the centrifugal force, the centrifuge speed, and the processing time [44]. Table 1 summarizes the differences between buffy-coat-based PRP and plasma-based PRP technologies.

	Buffy coat-based PRP	Plasma-based PRP
Performance and technology features
High volume preparation devices	++++	−
Double spin protocols	++++	+
Platelet capture rate %	++++	+
PRP volume range	++++	+
Platelet dosing strategies	++++	−
Flexibility in bioformulation	++++	−
Sustained release feature	++	++++
Cellular Features
Platelet growth factor concentrations	++++	+
Immunomodulatory capacity	++++	+
Angiogenetic properties	++++	+
Nociceptive effects	++++	−
Collagen synthesis	++++	+
Fibroblast proliferation	++++	+
Leukocyte Control	+++	+
PRP to plasma protein ratio	+	++++

Table 1.

Differences illustrated between buffy coat-based PRP and plasma-based PRP technologies regarding general device performance characteristics and cellular features of both technologies.

++++: highly feasible; ++: feasible; +: potentially possible; −: not feasible.

3.1.3 Platelet lysate preparations

Platelet lysates (PLs) are prepared following a PRP preparation procedure. As a result of freezing and thawing protocols, the membranes of PRP platelets are mechanically disrupted, allowing growth factors, cytokines, and other granular contents to be released. PLs are more than minimally manipulated ABPs, and they are a source of acellular, platelet-free, biologically active supernatants, which are rich in PGF and cytokines, as well as angiogenic factors. Additionally, as with PRP preparations, any concerns regarding immunogenicity can be dismissed as well [45]. The mechanisms of action of PL are poorly understood, but theoretically, there should be a similar mechanism of action between PL and PRP [46, 47]. Nonetheless, PGFs present in PLs have a short half-life, and they are reported to negatively affect endothelial cell proliferation as well as vascular tube formation in in vitro research [48].

The growth-promoting properties of PLs have been studied in multiple clinical indications, including wounds and soft tissue injuries, as early as the 1980s. A renewed interest in PLs has led to their use in specific musculoskeletal pathologies [49], in part due to their alleged anti-inflammatory properties [50]. Many advocates of PLs assert that their main advantage is that they can be stored in a freezer as well as that they are readily available for subsequent procedures or future applications [51].

Like PRP, there is neither consensus on the preparation of PL protocols nor is there a clear indication for its use, despite some reports citing PL applications in intra-articular procedures and tendinopathies, and in lumbar radiculopathies [50, 52].

4. MSC-based biological preparations

Adult MSCs are undifferentiated multipotent cells that are found in virtually all organs of the human body, and they are known for their self-renewal capacity. Easily accessible tissues to harvest MSCs include the red bone marrow and adipose tissue SVF [5]. In addition to their potential to promote immune modulation, angiogenic activity, anti-inflammatory activity, and anti-apoptotic activity, they also release numerous cytokines and growth factors that confer immunomodulatory, anti-apoptotic, and anti-inflammatory effects on many heterogeneous populations of cells [9]. Furthermore, MSCs have the ability to differentiate into various cells of mesodermal origin, including adipocytes, chondrocytes, myocytes, and osteoblasts, when exposed to specific signaling pathways [53].

Density centrifugation techniques are used to concentrate aspirated bone marrow and lipoaspirates to produce bone marrow aspirate concentrate (BMAC) and tSVF, respectively. Unfortunately, similar to PRP preparations, there is no consensus on classification, preparation standards including validation, and quality controls.

Adipose-derived MSCs (AD-MSCs) and BM-MSCs share many biological characteristics [54]. Several studies indicated that MSC transcriptome profiles from AD-MSCs and BM-MSCs showed the highest homology [55]. Although some differences in their immunophenotype, differentiation potential, proteome, and immunomodulatory activities have been reported [56]. Additionally, some clinicians have been reporting on using bone marrow aspirate (BMA) as an ABP, in particular in musculoskeletal pathologies [57].

4.1 Bone marrow aspirate: aspiration and direct injection

Cortical and trabecular bones, cartilage, and connective tissues comprise the bone. The bone marrow tissue, like the peripheral blood, is composed of soft spongy bone, fat-rich marrow, and sinusoidal arterial and venous blood vessels. Bone marrow is a life tissue as it is well recognized as an abundant and heterogeneous source of cells that reside in the trabecular part of long bones and are ultimately, continuously, released into the bloodstream [58].

Bone marrow aspirate (BMA) can be minimally invasive extracted from various anatomical sites using simple bone marrow harvesting needles or more sophisticated bone marrow aspiration systems. The posterior superior iliac spine (PSIS) and anterior superior iliac spine (ASIS) are frequently cited harvesting sites since these sites have the highest MSC concentration among other anatomical sites [59].

BMAs are ABPs with the intent to use an aspirate-collecting device only. In general, a single 10 ml syringe is used to extract bone marrow followed by direct administration of the harvested BMA to pathological treatment locations [57]. Using BMA only is intentionally done to avoid filtration techniques and BMA concentration processing steps, thus avoiding any methods to increase all native cell counts in the treatment specimen [60]. Only few articles have been published in the sports medicine and orthopedic literature regarding BMA applications [61]. Rarely, direct comparisons between BMA and BMAC have been executed and clinical benefits of BMA injections have not been clearly elucidated [60].

4.2 Bone marrow aspirate concentration: BM-MSC preparations

In contrast to BMA treatment vials, BMAC is prepared from a particular harvested volume of BMA, using gravitational density centrifugation processing steps, similar to PRP preparations [62].

BMAC contains concentrated bone marrow MSCs (BM-MSCs), hematopoietic stem cells (HSCs) as well as their multipotent progenitor cells (MPCs), platelets, leukocytes, and myeloid cells. Distinct differences between a BMA and a BMAC treatment specimen is that the latter has significantly more bone marrow cellular concentrations, and platelets when compared to BMA [60]. Importantly, the RBC content in BMAC is significantly lower due to the use of bone marrow tissue fragmentation as a result of centrifugation, decreasing the RBC concentration and thus avoiding all the negative effects related to the presence of native RBCs concentrations [63].

The cellular composition of BMAC is more complex and distinct when compared to PRP, as it contains BM-MSCs and HSCs, each with its own MPCs, which provide distinct reparative functions, other than concentrated PRP.

Prior to BMA harvesting, it is important to recognize that the bone marrow cavity is a well shielded cellular environment consisting of a particular structure and segmented in 4 main regions, niches, according to Lambertsen and Weis [64]. A bone marrow niche is a dynamic and complex environment, defined by its specific cellular content and molecular environment to regulate the function of stem cells and progenitors. Furthermore, the three-dimensional niches contain cells capable of intercellular communications and interactions with the extra cellular matrix (ECM). Cells present in bone marrow niches define stemness and regulate stem cell renewal and quiescence through cell signaling, proliferation, and differentiation [65]. In bone marrow niches, newly formed cells are retained until they mature and are then released into the peripheral circulation [66].

The vast majority of BM-MSCs are found within the endosteal and subendosteal regions of the bone marrow entity [67, 68]. Furthermore, BM-MSCs can be presented as pericytes in the perisinusoidal region [69]. As opposed to BM-MSCs, HSCs are located in the deeper bone marrow regions in the perivascular and perisinusoidal regions, and in the megakaryocyte niches. Most niche-specific processes are regulated by complex biochemical and mechanical signaling processes, followed by direct cell-to-cell interactions with platelet-derived chemokines, stem cell factors, and transforming growth factor [70].

BM-MSCs induce the production of cell-specific antigens, and their progenitors may differentiate into cells of the mesodermal lineage including osteoblasts, chondrocytes, tenocytes, endothelial cells, myocytes, fibroblasts, nerves, and adipocytes [71]. The differentiation of HSCs and progenitor cells into specialized blood cells plays a vital role in hemostasis, homeostatic mechanisms, and wound healing and angiogenesis in indirect ways [4]. Due to these reasons, BMAC has been regarded as an effective ABP to treat numerous clinical pathologies by altering local microenvironmental conditions through immunomodulatory activities, angiogenesis, and tissue repair.

4.3 Adipose tSVF based preparations

Adipose tissue preparations have been successfully used in minimally invasive reconstructive procedures and in guided injections into damaged, degenerative, or non-healing wounds. The rational to use tSVF is that it is a heterogeneous ABP, highly vascularized and stable connective tissue due to cell-to-cell or cell-to-matrix connections which are considered a pre-requisite for such stem cells to open the cellular send and receive signaling [72]. ATC, with its important bioactive scaffolding capabilities should therefore be recognized as a multifaceted microvascular organ, or better as an ATC, in the form of lipoaspirates, consisting of various cellular tissue components, including AD-MSCs [73], Furthermore, concentrated and compressed tSVF provides a physiological multicellular scaffold containing AD-MSCs along with other stromal vascular cells [19]. Minimally invasive access techniques are employed to harvest from several subcutaneous fat deposits, including the abdomen, perigluteal, thigh, and flank areas. During the procedure, a subcutaneous injection of a dilute local anesthetic solution (tumescent) is administered to numb the area of adipose harvesting and facilitate the creation of a tSVF suspension [74]. After a brief waiting period, a blunt-tipped atraumatic aspiration cannula is introduced through a small puncture or incision in the skin and attached to a syringe for manually controlled, low negative pressure, lipoaspiration [75]. A correctly performed lipoaspiration procedure preserves the neurovascular structures at the donor site with a minimal level of discomfort for the patient [76]. Furthermore, the cell yield and viability of AD-MSCs should not be affected by liposuction techniques [77, 78].

Harvested tSVF can be transferred to a syringe or adipose processing device and centrifugated. Centrifugation methods have proven to be an effective means to safely wash, rinse, eliminate the infranatant-extracellular fluid, separate free lipids, and residual oil, to prepare a clean, compressed tSVF specimen [79], as illustrated in Figure 6.

Figure 6.

An illustration following adipose tissue centrifugation to access tSVF (A). After centrifugation, first the residual oil (B) and then the infranatant solution (C) are removed, thereafter the tSVF is extracted (D). (Device shown in the picture is the Progenikine® Adipose Concentration System EmCyte Corporation®, Fort Myers FL, USA).

Following lipoaspirate centrifugation, isolation of tSVF-based preparations can be accessed, consisting of cellular and matrix components derived from adipose tissue and are used in advanced regenerative medicine procedures. The SVF is a complex mixture of various cell types including endothelial cells, immune cells, fibroblasts, and a significant population of AD-MSCs. These cells are embedded in a network of collagen fibers and other extracellular matrix components, which together contribute to the regenerative properties of the SVF.

Prior to regenerative applications, various SVF preparation protocols are obtainable to prepare either fully emulsified nanofat in small aggregates (tSVF) or cSVF [80]. cSVF preparation methods are based on enzymatic digestion techniques using collagenase [81]. tSVF preparations are created by mechanical emulsification, reducing the size of macrofat to either partially or fully emulsified aggregates, known as micronized fat or nanofat (Figure 7) [80]. Importantly, the fully emulsified, sized tSVF aggregates present an intact microenvironment, whereby the fully emulsified small nanofat aggregates essentially are devoid of adult adipocytes. Generally, nanofat tSVF preparations are considered the most potent valuable form of tSVF for regenerative and wound healing purposes.

Figure 7.

Mechanical emulsification systems. In A, “partial emulsification” is realized by shifting the syringes back and forward through restraining female-female luer connectors to create micronized tSVF. In B, the micronized tSVF is ready for application. In C, adipose tissue is pushed through a 600/400micron screen, creating nanofat tSVF (devices shown are in A the Adicen™, a three phase micro-fragmentation accessory with 2.4, 1.4, and 1.2 mm emulsification sizes, EmCyte Corporation®, Fort Myers FL, USA. In C, the disposable Nanofat™ device is displayed, Tulip® Medical, San Diego CA, USA).

4.3.1 Multicellular SVF components

The presence of SVF-based cells can be tested by enzymatic means and measured by advanced laboratory flow cytometry techniques. The typical, heterogenous cell distribution and the cellular ranges in SVF are shown in Table 2 [82].

Stromal Cells:	15–30%	AD-MSC
		Pre-adipocytes
		Fibroblast
Stem cell and progenitors	< 0.1%
Hematopoietic cell linages:		Platelets
		Erythrocyte
		Lymphocyte
		Neutrophil
		Monocyte – macrophage
Pericytes	3–5%
Endothelial cells	10–20%

Table 2.

Overview of identified multicellular components in SVF.

AD-MSCs are the most prominent stem cells in tSVF. They have the capability to differentiate into various cell types, including osteoblasts, adipocytes, chondrocytes tenocytes, and myocytes. Many researchers and clinicians believe that pericyte stem cells (PSC) are ubiquitous cells, representing the original “stem” cell originator [83] and are fully capable of all the functions of the MSCs. PSCs are located on the walls of capillaries, the perivascular/paravascular locations, and work intimately with the endothelial and intra-adventitial cells. Furthermore, PCS stabilize blood vessels and have the potential to differentiate into other cell types, contributing to tissue regeneration and repair. Endothelial cells can line blood vessels and play key roles in forming new blood vessels through angiogenesis. Immune cells, originating from hematopoietic cell linages (e.g., monocytes, lymphocytes, and various macrophage phenotypes) are involved in modulating the immune response. Lastly, fibroblasts are present in SVF and they are recognized for producing collagen and an extracellular matrix, providing structural support to tissues and they hold crucial roles in wound closure and the strength of the healed tissues [84].

Freshly prepared SVF can be directly administered to the patient without the need for cell expansion techniques or additional cell separation preparations. Unlike cSVF, emulsified tSVF is not solely a 100% cellular product, as it contains both cellular fragments and important native adipose structural ECM components) [80, 85, 86]. Following tSVF preparation, the product is ready for injection. An advantage of tSVF compared to cSVF is that it offers the ability to provide a bioactive cellular tissue matrix that can be delivered to pathoanatomic treatment sites. In fact, following tSVF application, stromal supportive cells in their microenvironment are being transferred to recipient sites where these stromal elements start cell-cell communication and cell signaling [87].

4.3.2 The role of tSVF in wound healing, repair, and regenerative therapies

The application of tSVF in wound healing and regenerative therapies are based on its rich content of regenerative cells and factors. tSVF cells are capable of differentiation, cell-to-cell communication (paracrine, sending/receiving when attached, and may secrete ECM, and provide key elements in the response to damaged or degenerative cells [73]. tSVF aids in the promotion of angiogenesis as endothelial cells and growth factors promote the formation of new blood vessels, enhancing oxygen and nutrient supply to the wound or damaged tissue, thereby supporting healing [88]. Immune cells and anti-inflammatory cytokines present in tSVF support in modulating the body’s immune response to reduce chronic inflammation while promoting a more conducive environment for tissue repair. Following tSVF preparation, the AD-MSCs can self-replicate and they are capable of differentiating into various cell types, secrete growth factors, help with creation of biologically active ECM, and may provide mesodermal, ectodermal, neuroectodermal cells that contribute and stimulate tissue regeneration [89]. This is particularly valuable in tissue repair and regeneration. Finally, fibroblasts contribute to the synthesis of collagen and other extracellular matrix components, essential for the structural integrity and function of repaired tissues [90].

5. Combined ABPs

PRP, BMAC, and tSVF are ABPs that contribute to immunomodulation, painkilling, wound healing, and functional tissue restoration with different intensities and distinctly different pathways.

Many studies have reported on the beneficial effects of PRP and MSC-based preparations for the treatment of acute cutaneous and chronic wounds, degenerative tissues, and burns [91, 92]. Some of the therapeutic effects of PRP are recognized by the release of the platelet granular content, following in-situ platelet activation [10]. Platelet have been found, among other activities, to stimulate fibroblast proliferation and differentiation and incite endothelial cellular pathways regarding angiogenesis and ECM secretion to regulate tissue inflammation [93]. In addition, following platelet activation, the PRP fibrin network provides an autologous cellular scaffold [6].

Individually, BM-MSCs and AD-MSCs have demonstrated their cellular differentiation potential and immunomodulatory effects. Not many studies have addressed the combination of both MSCs, but they are routinely being performed in regenerative medicine center. Wardner et al. concluded that the proliferation of peripheral blood mononuclear cells was more suppressed by AD-MSCs, however, combining them with BM-MSCs resulted in highly efficient immunomodulation, with significant responder cell suppression in a dose dependent manner, possibly reducing the inflammatory effects when BMAC is used as a stand-alone product [94].

The delivery of MSCs exert tissue repair effects primarily through the release of a wide range of soluble factors, growth factors, cytokines, and exosomes capable of proangiogenic and anti-inflammatory performances [95, 96, 97]. In contrast to PRP, both BM-MSCs and AD-MSCs can modify their cellular secretome according to the local microenvironment in which they have been injected or applied and maintain cell-signaling to neighboring cells for several days [98].

Based on the typical PRP, MSC, and plasma protein characteristics, it was suggested that potentially positive synergistically effects may occur if PRP is combined with BM and AD-MSC preparations. Additionally, PRP effects might be extended over a longer period of time when mixed with plasma protein concentrates and consolidated as one ABP [99].

5.1 Protein rich-platelet rich plasma (PR-PRP): concentrated platelet poor plasma mixed with PRP

PRP buffy-coat preparations are created by gravitational density separation that sequesters a unit of blood into three main fractions: a PPP fraction, a buffy coat stratum rich in platelets, and a RBC fraction. Normally, as part of the PRP preparation process, the PPP fraction is discarded after the platelet concentrate has been extracted from the PRP device.

One of the less well known, but equally important features of PPP is the presence of exosomes and other macrovesicles [100, 101]. They are considered to be important in cell-cell communication and cell signaling. Furthermore, some GF are not present in the platelet alpha granules, but instead, they reside in higher concentrations in the PPP fraction, outside of platelets. In particular insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF).

In addition to their roles as angiogenesis activators, these plasma-based GFs are also known to promote keratinocyte migration and wound healing. Interestingly, Beitia et al. found a significant correlation between IGF-1 concentrations and cell proliferation [102]. Moreover, it has been shown that both IGF-1 and HGF can inhibit inflammation and fibrosis.

Technology advancements in ABPs have led to the development of more advanced biological processing methods and devices. By using new ultrafiltration technologies, it is possible to concentrate PPP, at point-of-care, in a fast and efficient manner producing a viable and viscous protein concentrate [103].

Microfiltration and ultrafiltration devices are made of high-performance medical grade polymers and offer an excellent biocompatibility and present a consistent performance eliminating plasma water. They have been used widely as blood-plasma contact devices in blood apheresis for decades [104]. Later, modified ultrafiltration techniques were successfully developed to alleviate systemic inflammatory responses caused by cytokines [105].

Currently, ultrafiltration techniques can incorporate PPP processing methods. The PPP fluid fraction is manually transported by syringes, exerting a positive transmembrane pressure, through the microporous membranes of the ultrafiltration device fibers. Once the PPP volume has frequently passed the ultrafiltration device, plasma water, cytokines, molecules, and plasma proteins with a molecular mass (weight), less than the pore size of the fibers, are all filtered out of the ultrafiltration device. This waste solution is collected in an effluent syringe, as demonstrated in Figure 8.

Figure 8.

The platelet-poor plasma (PPP) syringes are manually “pushed” through the ultrafiltration device (A). Plasma water, small proteins, and cytokines are eliminated through the filter membrane pores and captured in the effluent syringe. After a series of passes, the PPP volume is significantly reduced to a high viscous protein rich PPP volume, as shown in B. Note the clear visual difference between the unprocessed PPP and the final viscous protein rich PPP (the ultrafiltration device presented is the CORE™ Ultrafiltration System, developed by EmCyte Corporation®, Fort Myers FL, USA).

Reducing the PPP water component will result in a significant increase in the concentration of, functional, total proteins, like fibrinogen, albumen, and alpha-2-macroglobulin. Moreover, plasma levels of IGF-1 and HGF are also significantly higher after ultrafiltration when compared to their levels in the native PPP fraction before the filtration process [106]. Effectively, larger non-concentrated PPP volumes will be reduced to low volume protein rich PPP, up to 10-fold reductions can be achieved.

Recently, EmCyte Corporation® has introduced the concept of CORE™ protein rich platelet concentrate formulation. This consolidated ABP, combining a high concentration of PRP platelets with a high concentration of plasma proteins (Figure 9), has distinctive scaffolding features, protease inhibitor, and regenerative capabilities when compared with other similar ABPs on the market.

Figure 9.

PR-PRP preparation. A low volume of protein rich PPP (left syringe) is cautiously mixed with the same volume of highly concentrated PRP (right syringe) creating PR-PRP, at a ratio of 1:1. Before application, the consolidated volume is gently agitated to produce a uniform ABP treatment specimen. Abbreviations: PRP: platelet-rich plasma; PPP: platelet-poor plasma; PR-PRP: protein-rich platelet concentrate; ABP: autologous biological preparation.

The creation and ultimate delivery of PR-PRP in tissue repair, regeneration, and wound healing procedures can be considered as a novel and powerful therapeutic.

Depending on the bioformulation of the prepared PRP, the PR-PRP scaffold retains and facilitates interactions between a variety of concentrated heterogeneous PRP components and molecules: activated and non-activated platelets, PGFs, dense platelet granular elements, plasma-based IGF and HGF, cytokines, plasma proteins, exosomes, and eventually leukocytes [107], as presented in Figure 10.

Figure 10.

A graphic illustration of a PR-PRP scaffold with the retained platelets, and leukocytes. Interactions between tissue plasminogen (tPA) and plasminogen (PA) binding to the scaffold with the subsequent production of plasmin (P) to accomplish fibrinolysis, leading to fibrinolysis with the release of the retained PR-PRP elements. Abbreviations: tPA: tissue plasminogen; PA: plasminogen; P: plasmin.

The use of PR-PRP preparations in non-surgical interventional procedures in musculoskeletal pathologies includes, among others, intra-articular injections in patients with OA, and patients with partial of full-thickness tendon tears. In OA, the PR-PRP specific protein alpha-2-macroglobulin (A2M), a macro protein molecule known to function as a broad-spectrum protease inhibitor, traps active joint proteases into the A2M typical molecular cage and subsequently eliminates them from the joint space [108]. In patients with partial or full thickness tendon and ligament tears, the PR-PRP proteins fibrinogen and albumen function as a biological active tissue scaffold, capable of filling tissue voids [109, 110]. Additionally, the scaffold facilitates a sustained platelet release of PGFs, cytokines, molecules, and eventual leukocytes, stimulating the local environment for a longer period to regulate inflammation, immunomodulation, and extend the tissue repair processes.

In chronic wound care management, autologously prepared PR-PRP grafts have been used as biological wound dressings [5, 111]. The wound healing process is meticulously controlled by various cytokines and growth factors that are secreted to the wound area [112], with platelets holding pivotal roles in wound healing by platelet plug formation, PGF release and the initiation of the angiogenesis cascade [14]. Platelets in PR-PRP matrix serve as vital scaffolds where platelets release PGF, cytokines, molecules, and other granular content that facilitate wound closure by acting as chemotactic cues to inflammatory cells and stimulate angiogenesis. In the protein matrix, the presence of thrombin facilitates platelet degranulation with the subsequent release of platelet content. Furthermore, concentrated plasma proteins such as thrombospondin, vitronectin, and fibronectin are important in cell adhesion and cell migration in the wound healing cascade [113]. Ultimately, these processes can result in wound re-epithelialization and wound contractions of connective tissue. PR-PRP can either be injected in wound tissue structures, or it can be prepared and activated in a petri-dish and applied as a biological wound dressing to open chronic wounds, as demonstrated in Figure 11 [114].

Figure 11.

Different PR-PRP application modalities can be prepared to treat, open, chronic wounds. In A, a leukocyte-rich PR-PRP is administered as a topical and liquid ABP to a chronic leg ulcer. In B, a leukocyte-poor PR-PRP is prepared in a petri-dish as a solid biological graft which can be applied to chronic wounds as an autologous biological wound dressing.

PR-PRP spray applications can be effectively used during surgical interventions, referred to as biosurgery, to support in post-operative wound healing (Figure 12). Specifically, the combination of PRP characteristics with protein rich fibrin scaffolding in a variety of biosurgical procedures, are effective in reducing dehiscent wounds, lowering infection rates, reducing scarring, as well as reducing seromata formation, thus improving overall patient outcomes [2, 3, 115, 116, 117, 118].

Figure 12.

An illustration of PR-PRP applications in orthopedic (A) (total knee replacement) and plastic reconstructive (B) (lower body lift) biosurgical procedures. A dual syringe spray applicator is used to deliver the combined PR-PRP preparation and mix it with thrombin to support in post-surgical wound healing. Both, leukocyte-rich and leukocyte-poor PRP formulations can be combined with the protein concentrate. Abbreviations: PR-PRP: protein rich platelet concentrate; LP-PRP: leukocyte-poor platelet-rich plasma; LR-PRP: leukocyte-rich platelet-rich plasma.

After the PR-PRP scaffold has been delivered to tissue sites, the protein scaffold will start to breakdown due to the interaction of tissue plasminogen (tPA) with the inactive enzyme plasminogen (PA). Subsequently, PA will bind to the PR-PRP and will release the catalytically active nonspecific protease plasmin [119]. The proteolytic plasmin initiates plasma hydrolysis of cross-linked fibrin polymers within the scaffold [120]. In response, the PR-PRP scaffold will undergo a fibrinolytic breakdown (fibrinolysis), thereby releasing the cells that have been retained in the PR-PRP scaffold. A sustained release of concentrated cells and molecules in the PR-PRP scaffold over a longer period will transpire until the entire scaffold has been dissolved following fibrinolysis.

Future studies have to elucidate that this biologically created, sustained release-based, microenvironment holds an important role in tissue regeneration, immunomodulation, and ultimately functional tissue repair.

5.2 PRP and BMAC combined

Several studies have been indicating positive effects of combining PRP and BMAC as one consolidated ABP [121, 122, 123, 124], although cellular and cytokine characterization revealed overlapping data [125]. While interleukin-1 receptor antagonist (IL-1RA), a potent natural anti-inflammatory cytokine, was significantly increased in BMAC when compared to PRP [126]. Combining PRP and BMAC may create a more biologically active graft, projected to optimize tissue repair, regeneration, and wound healing treatment outcomes as concentrated MSCs and HSCs are added to the PRP cellular components, including significantly more anti-inflammatory activity due to higher concentrations of IL-1RA (Figure 13). An analysis concluded that PRP stimulates BM-MSC proliferation and preserves MSC multipotency, as the interactions between PRP and MSCs did not affect multilineage cell differentiation [99]. Additionally, PRP had a positive effect on MSC senescent activities. In an in vivo ACL study, the combined stimulatory effects of PRP and MSCs demonstrated improved MSC proliferation, cellular growth, and ligament integrity [127].

Figure 13.

A graphical comparison between leukocyte-rich PRP (LR-PRP) (A) and BMAC (B) cell distributions. When consolidating LR-PRP with BMAC, this ABP is characterized by high concentrations of platelets, BM-MSCs, and BM-HSCs. Noteworthy, the plasma color of the BMAC preparation is more orange than the yellow appearance of the LR-PRP preparation. This typical colorization aspect of a BMAC preparation is related to the higher shear forces employed when extracting bone marrow to liberate the BM-MSCs from their specific niches. Abbreviations: PRP: platelet-rich plasma; HSCs: hematopoietic stem cells; BM-MSCs: bone marrow mesenchymal stem cells; BMAC: bone marrow aspirate concentrate.

According to a review, citing 57 articles, studies using PRP showed a reduction in the time necessary to double the number of BM-MSCs [99]. A further study showed that MSCs cultured with PRP instead of fetal bovine serum altered the secretion of cytokines by T lymphocytes, B cells, and natural killer cells, demonstrating significant anti-inflammatory activities. In an animal study, the combination of PRP and BM-MSCs demonstrated an increase in the maturation of trabecular bone along with an increase in collagen type II expression and differentiation to chondrogenesis [128].

Improved VAS and functional scores were observed following combined PRP-BMAC injections in patients with partial rotator cuff tears, when compared to control patients [124]. It was thought that the PRP-BMAC consolidated biologic was responsible for the associated pain killing effects by downregulating various inflammatory mediators [127, 129].

PRP-BMAC complexes have revealed that PGFs and other platelet cytokines are influential in the BMAC reparative initiated processes as they stimulate multiple paracrine BM-MSC trophic effects, including endogenous tissue repair, while decreasing cell apoptosis expressing and releasing higher concentrations of therapeutic affecters [124, 125]. Additionally, a decrease in cell apoptosis and inflammation has been reported. Besides, combining PRP and concentrated BM-MSCs with a demineralized bone matrix (DBM) guaranteed the consistent release of high PGFs over time in an in-vitro model [130]. It was postulated that the clinical application of a PRP-BMAC graft in a bone matrix will allow for better healing in compromised tendons [98]. In another study, the response of BM-MSCs was measured in healthy human tendons, DBM, and a fibrin scaffold. MSC stromal cell adhesion, proliferation and differentiation assays indicated differences in cellular activity among the three different scaffolds. However, when PRP was added to the BM-MSCs, enhanced cellular function was observed in all three scaffolds, indicating significant synergistic effects by the combined PRP-BMAC biological preparation [131].

5.3 PRP and tSVF combined

The effectiveness of PRP and SVF modalities as biological treatment options for a variety of pathological conditions has been studied in numerous clinical trials.

Recently, compounding PRP and tSVF as a combined ABP (Figure 14) have been used by practitioners in mainly esthetic and plastic reconstructive surgical procedures, and to treat patients with non-healing chronic wounds. The rationale behind this combination is to increase the survival rate of fat grafts, to decrease inflammation, and ultimately improve long term fat grafting outcomes [133, 134].

Figure 14.

In A, LR-PRP is mixed with processed tSVF to create a compounded ABP. In A, the less dense adipose tissue is mixed with the denser PRP, insinuating that proper agitation is required to create homogenously mixed graft prior to application [132]. Ideally, multiples smaller application syringes (B) are prepared after mixing both the PRP and compressed tSVF volumes to ensure proper distribution of the PRP-cATC graft, targeting for significant synergistic therapeutic effects. Abbreviations: LR-PRP: leukocyte-rich platelet rich plasma; cATC: compressed adipose tissue complex.

PRP combined with AD-MSCs was shown to significantly stimulate tissue vascularization in wounds as compared to PRP or AD-MSCs alone [135]. Similar findings were reported by others [136, 137]. According to their findings, PRP stimulates the angiogenic potential of AD-MSCs by activating the MSC secretome directly, which results in a stimulation of the VEGF and SDF-1 synthesis, thereby improving blood vessel formation and endothelial cell migration. Others observed a synergistic effect between tSVF and the PRP induced fibrin matrix, with regard to the secretion of high VEGF concentrations [138]. Another important finding was that PRP promoted the proliferation of AD-MSCs via various pathways [139]. Furthermore, van Dongen et al. concluded that the combination of PRP growth factors and the mechanically dissociated, emulsified ATC (tSVF) are proficient in parenchymal cell proliferation, and immunomodulation [140].

Intriguingly, PRP combined with AD-MSCs increased mitochondrial respiration and oxygen consumption, resulting in an increase in ATP production. The addition of PRP to AD-MSCs increased the AD-MSC metabolism and fat graft vascularization, ultimately resulting in improved fat graft survival and stimulation of the recipient tissue site [134]. Importantly, when PRP was mixed with adipose grafts, a reduction in inflammatory reactions, as well as a reduction in fat necrosis percentage was observed [141, 142, 143]. Synergistic cellular and GF specific effects, after mixing PRP with an adipose tissue graft, are presented in Table 3.

	Vascularization	Proliferation; Osteoblasts	Proliferation Fibroblasts	ECM; Synthesis	Chemotaxis
PDGF	*	++	++	++	+/−
TGF	*	+/−	+/−	++	+
VEGF	++	*	—	+	—
CTGF	++	+/−	++	+	—
IGF	—	++	+	++	++
Fibrin	++	—	—	++	—

Table 3.

The synergistical and contributing dynamics of specific PRP growth factors and plasma-fibrin fraction to adipose tissue grafting cellular processes.

*: indirect effect; +: increased; ++: highly increased; −: no effect [136, 144, 145, 146, 147].

Abbreviations: ECM: extra-cellular matrix; PDGF: platelet-derived growth factor; TGF: transforming growth factor; VEGF: vascular endothelial growth factor; CTCG: connective tissue growth factor; IGF: insulin-like growth factor.

It is important to note that after mixing lipoaspirate formulations with PRP, a PRP-tSVF-fibrin scaffold will develop as a combined product. This scaffold serves as a cellular matrix and retains adipocytes, AD-MSCs, PGFs, cytokines, and other proteins for an extended period of time, serving as a sustained, extended-release medication [148]. In addition, it has been shown that the PRP-tSVF-fibrin scaffold inhibits apoptosis of adipocytes [149].

Despite some initial positive reports in chronic wound healing models [147], additional clinical research is needed to develop more synergistic formulations of ABPs and application strategies for the treatment of degenerative conditions, poorly healing chronic wounds, as well as to support in patient outcomes following biosurgical procedures.

6. Conclusions

ABPs, prepared with modern and dedicated devices and ultimately precisely delivered, are powerful high yielded cellular biological products.

The employment of properly prepared stand-alone or combined biologics is a valuable and safe therapy for clinicians to repair and regenerate tissues, aiming for functional restoration. Furthermore, they can be used as supportive armamentarium in wound healing treatments and biosurgical procedures to improve patient outcomes and decrease complications, as evidenced by an extensive body of clinical literature.

Lastly, we believe that more clinical and translational studies, as well as educational activities, are required in order for ABPs to be widely accepted as mainstream therapeutics.

Conflict of interest

Other authors declare no conflict of interest.