Platelet-rich plasma and Bone Marrow Aspirate Concentrate in Wound healing

INTRODUCTION
Wound healing is a complex and dynamic process. Once a wound begins healing, normally the process resolves with complete wound closure. However, healing of acute and chronic wounds can become impaired by patient factors (ie, comorbidities) and/or wound factors (ie, infection). Restarting a wound with impaired healing is difficult because good standard wound care does not always provide an improved healing outcome and often more advanced therapies are employed.

Platelet rich plasma (PRP) gel is considered to be advanced wound therapy for chronic and acute wounds. For more than 20 years, PRP gel has been used to stimulate wound healing. Autologous PRP gel consists of cytokines, growth factors, chemokines, and a fibrin scaffold derived from a patient’s blood. The mechanism of action for PRP gel is thought to be the molecular and cellular induction of normal wound healing responses similar to that seen with platelet activation.

PRP AND ITS CLINICAL APPLICATION IN WOUND HEALING

From a historical perspective, PRP application started in regenerative medicine the 1980s. At the end of the 1990s, taking advantage of the sealing and hemostatic properties of fibrin, PRP was progressively used in oral and maxillofacial medicine. After the first description of an ambulatory method for PRP obtainment by Anitua in 1999, various techniques and potential uses have been described. At present, different PRP cell-separation systems are commercially available.

 

DEFINITION
PRP is a biological product defined as a portion of the plasma fraction of autologous blood with a platelet concentration above the baseline (before centrifugation). As such, PRP contains not only a high level of platelets but also the full complement of clotting factors, the latter typically remaining at their normal, physiologic levels. It is enriched by a range of GFs, chemokines, cytokines, and other plasma proteins.

The PRP is obtained from the blood of patients before centrifugation. After centrifugation and according to their different density gradients, the separation of blood components (red blood cells, PRP, and platelet-poor plasma [PPP]) follows.

In PRP, besides the higher concentration of platelets, other parameters need to be taken into account, such as the presence or absence of leucocytes and activation. This will define the type of PRP used in different pathologies.

There are several commercial devices available, which simplify the preparation of PRP. According to the manufacturers, PRP devices usually achieve a concentration of PRP 2-5 times the baseline concentration. Although one might think that a higher platelet count with a higher number of GFs would lead to better results, this has not been determined yet. Concentration of PRP 2.5 times above the baseline could have an inhibitory effect.

The most commonly used technique is to obtain a blood simple from the patients themselves (autologous), but homologous techniques are also a valid option. The blood is centrifuged to separate the platelets from red and white blood cells. Depending on the author, single or double centrifugation under different centrifugation times and speed conditions may be used. The objective is achieving highly concentrated platelets and suspended in a small volume of plasma, which is consequently rich in growth factors. The mean blood platelet count in normal individuals ranges from 150,000 to 350,000/μL. Although a PRP platelet count of 1 million/µL (baseline levels ×5) has been postulated as the ideal therapeutic dose of PRP, others propose that platelet integrity is more important than platelet concentration and suggest that PRP should be defined as the volume of plasma that has more platelets than baseline blood.

METHOD
When treating a lesion with PRP, the amount of bioavailable growth factors depends on both the platelet storage and the release into the microenvironment. This release relies on the kinetics of uptake and release from the PRP. Few studies have compared the kinetics of growth-factor release among PRP gels obtained by different methods. Differences are associated with platelet damage secondary to manipulation, variety in fibrin-mesh characteristics, which depend on the procoagulant molecule that has been used (calcium or thrombin), and growth factor-dependent factors. For example, ex vivo thrombin-activated PRP induces rapid clot formation/retraction and a sudden rise in molecular signals compared with Ca2+ or collagen.Considering that clotting cascade can be activated in situ without an exogenous activator, some methods use nonactivated platelets on the basis of potentially more efficient stimulation of wound healing.

PRP application in wounds may be intralesional or topical. No studies compare the effects of both methods. Intralesional application will be limited by ulcer extent and patient pain tolerance. In our experience, considering pain tolerance, intralesional application of PRP should be restricted to small or neuropathic wounds.PRP injection should take place within the first 10 minutes after PRP activation with the procoagulant substance. PRP will be injected in wound edges in the wound bed. Topical application may be combined with intralesional use.

As happens with wound dressings and the different alternatives and advanced treatments, efficacy will mainly depend on the presence of a properly prepared wound bed. Prior to PRP application, the wound should be cleaned and adequately debrided. If there exists a high amount of necrotic or unviable tissue, it should be removed and PRP postponed until necessary. The choice of the secondary dressing depends on the amount of wound exudate. No studies have established the most appropriate frequency of PRP application. However, it is normally used on a weekly basis.

GROWTH FACTORS
As mentioned previously, platelets have the capability to release the wide variety of proteins they contain in differential patterns of spatial and temporal delivery. This is particularly important when considering the process of wound healing as a continuum. Three overlapping phases characterize cutaneous wound healing: (1) inflammatory, (2) proliferative, and (3) remodeling. Following injury platelets respond by forming the hemostatic plug to prevent further bleeding. Through secretion of factors of coagulation a fibrin rich clot is made. This serves as the provisional matrix to support cell proliferation and migration.

Platelets begin to secrete active factors within 10 min of forming the clot. Activation, or degranulation, leads to the fusion of granules with the plasma membrane and expulsion of the contained proteins into the local environment. Secreted growth factors and cytokines diffuse into the environment for the recruitment of neutrophils and macrophages and the stimulation of resident stem cells, endothelial cells, osteoblasts, fibroblasts, and epidermal cells.

These factors include platelet-derived growth factor, stromal-derived factor-1 alpha, regulated on activation, normal T cell expressed and secreted, vascular endothelial growth factor, and transforming growth factor beta (TGFb). Bound to cell surface receptors, they result in the activation of intracellular signaling cascades that result in migration, proliferation, or differentiation. Platelets as immune “cells” In addition to their roles in hemostasis, thrombosis, and wound healing mediation, platelets also have important roles in the immune response.

Platelets have been known to interact and adhere to inflamed endothelium whereby they interact with circulating leukocytes to promote aggregation and migration. Following adhesion to endothelium and aggregation, platelets express toll-like receptors known to promote neutrophil extracellular traps capable of capturing bacteria contaminating a wound. Platelet receptors such as P-selectin are capable of binding P-selectin glycoprotein ligand 1 on leukocytes inducing the activation of leukocyte through a conformational change of Mac1, a b2 integrin, to an active state. This interaction mediates neutrophil rolling and promotes transendothelial migration independent of neutrophil adhesion to endothelial cells.

Functional consequences of these interactions contribute to a range of pathologic and physiologic conditions that result in both beneficial and detrimental outcomes. Acute lung injury, atherosclerosis, and inflammatory bowel disease are all examples of how platelets interacting with inflamed endothelium, propagate the inflammation response, and amplify the systemic response. Conversely, platelet interactions in the presence of bacteria or sterile inflammation have shown beneficial responses leading to decontamination and enhance wound healing. A more detailed description of the interaction of platelets with leukocytes is out of the scope of this review; however, it is important to consider this function in the overall participation of platelets and platelet concentrates in the wound healing process.

CLASSIFICATION

Several authors have tried to characterize and classify the numerous techniques available on the market in terms of preparation (centrifugation speed and use of anticoagulant), content (platelets, leucocytes, and GFs), and applications.

Although the literature about PRPs developed with all these contradictions, the need for a standardized terminology is of great importance. Thus, some classifications have been proposed to achieve a consensus terminology in the field of platelet concentrates.

Characterizing the type of PRP used (as a pure PRPin our study) will lead to a better understanding of PRP, and data available will be easier to sort and interpret. Furthermore, this terminology would serve as a basis for further research on the topic.

In 2009, Dohan Ehrenfest et al. proposed a classification of 4 main families of preparations following 2 principle parameters: presence or absence of a cell content (such as leucocytes) and the fibrin architecture:

  1. Pure PRP or leucocyte-poor PRP: the preparation obtained is without leucocytes and shows a low-density fibrin network after activation.
  2. Leucocyte and PRP: the preparations contain leucocytes and show a low-density fibrin network after activation.
  3. Pure PRF or leucocyte-poor PRF: preparations are without leucocytes and with a high-density fibrin network. Unlike pure PRP or PRP containing leukocytes, these products cannot be injected and exist in an activated gel form.
  4. Leucocyte-rich fibrin and PRF: products are preparations with leucocytes and with a high-density fibrin network.

Another classification based on the presence or absence of white blood cells, activation status, and platelet concentration, based on the coefficients of an increase in the platelet and leukocyte concentration in PRP compared to the whole-blood baseline, as well as on PRP activation.

The classifications were not consensual and there is still the intent to search a classification for PRP that could characterize the injected PRP in order to compare the efficacy of different studies.

An important point of discussion is that in the previous classifications, the authors did not take into account the final volume of the preparation, the presence or absence of red blood cells (RBCs) in PRP, and the doses of platelets in the final volume of PRP obtained.

In 2016 the DEPA classification (Dose, Efficiency, Purity, Activation) that focuses on the quantity of platelets obtained by the PRP kits as well as on product purity and on platelet activation prior to injection.

THE DEPA CLASSIFICATION IS BASED ON 4 DIFFERENT COMPONENTS:

  1. Dose of injected platelets: calculated by multiplying the platelet concentration in PRP by the obtained volume of PRP. According to the injected dose (measured in billions or millions of platelets), it should be categorized into (a) very high dose of injected platelets of >5 billion; (b) high dose of injected platelets, from 3 to 5 billion; (c) medium dose of injected platelets, from 1 to 3 billion, and (d) low dose of injected platelets, <1 billion.
  2. Efficiency of the production: corresponds to the percentage of platelets recovered in the PRP from the blood. It is categorized as follows: (a) high device efficiency, if the recovery rate in platelets is >90%; (b) medium device efficiency, if the recovery rate in platelets is between 70 and 90%; (c) low device efficiency, if the recovery rate is between 30 and 70%, and (d) poor device efficiency, if the recovery rate is <30% and corresponds to the relative composition of platelets, leucocytes, and RBCs in the obtained PRP.
  3. Purity of the PRP obtained: correlates to the relative composition of platelets, leucocytes, and RBCs in the obtained PRP. It is described as (a) very pure PRP, if the percentage of platelets in the PRP, compared with RBCs and leucocytes, is >90%; (b) pure PRP, between 70 and 90% of the platelets; (c) heterogeneous PRP, if the percentage of platelets is between 30 and 70%, and (d) whole-blood PRP, if the percentage of platelets in the PRP is <30% compared with RBCs and leucocytes.
  4. Activation process: if an exogenous clotting factor was used to activate platelets, such as autologous thrombin or calcium chloride.

Although this last classification is very complete, this quantification cannot be defined by the physician and should be registered in each CE medical device available for preparation of PRP.

SAFETY OF PRP APPLICATION IN WOUNDS

PRP can be considered a secure treatment. No adverse effects, such as increased risk of infection or hypersensitivity reactions, have been detected in clinical trials. Regarding oncogenic potential, when possible coincidences between carcinogenesis and the mitogenic pathways employed by growth factors have been evaluated, no evidence supports a possible tumoral triggering. Once a growth factor has joined its membrane receptor, intracellular signal cascades are activated, normal genetic expression is promoted, and different control mechanisms regulate this process. Growth factor overexpression could give rise to receptor mutation.

It may be assumed that therapeutic growth-factor concentrates in PRP could act more as promoters than as initiators of carcinogenesis, favoring the division and proliferation of previously mutated cells. In tumor cells, the presence of an excessively large number of normal growth-factor-receptor copies induces increased sensitivity to the corresponding ligands, which even at very low concentrations may stimulate the cells and induce proliferation. Moreover, tumoral cells are unable adequately to suppress the continuously generated mitogenic signals Consequently, as the use of growth factors is contraindicated in malignant wounds, a skin biopsy should be taken before starting PRP if malignancy cannot be completely excluded.

 

CLINICAL EVIDENCE OF PRP AS ADJUNCTIVE TREATMENT FOR CHRONIC WOUNDS

The first clinical application of platelet-derived preparations was in chronic leg ulcers. The lesions were covered with collagen embedded in platelet proteins. With this product, known as platelet-derived wound-healing formula, the formation of vascularized connective tissue was induced in these wounds. Since then, different platelet preparations have been tried for application in solution, gel, or by injection in wounds of different etiologies.

Reports correspond mainly to individual cases or case series, although pilot studies and clinical trials have also been conducted. The outcomes of the isolated cases and small series published are often spectacular. These reports show noteworthy variability in the size and etiology of the lesions, as well as the method for obtaining and applying PRP. Wound etiologies that have been treated with PRP include diabetic, pressure, or venous ulcer, surgical or traumatic wounds, and wounds of other etiologies.

CONCLUSION

Regenerative medicine in wound healing is a continuously innovative area. Chronic wounds that do not respond to conventional treatment are not rare, and thus constitute a real challenge for the clinician. PRP is being used as a new therapeutic option for different pathologies in the field of dermatology, such as trichology, wound healing, and cosmetic medicine. In this manner, understanding the biology and mechanism of action of this therapy should help clinicians in selecting a system that meets their specific needs for a given indication.

In addition, characterizing the type of PRP used will lead to a standardization of PRP, making it easier to sort and interpret available dataPRP represents a viable alternative treatment for recalcitrant chronic ulcers, whose efficacy has been demonstrated both in vitro and in vivo. However, stronger scientific evidence is required to support its potential benefit for use in chronic wounds. Even though several studies describe interesting results with PRP application in chronic ulcers, the absence of clinical protocols and guidelines is hindering the extension of use. As other published reviews have previously concluded, robust clinical trials are essential to determine the most accurate indications for use and the most recommended method of PRP obtainment and application.

BONE MARROW ASPIRATE CONCENTRATE IN WOUND HEALING

WHAT IS BONE MARROW CONCENTRATE?
Bone marrow is the soft, spongy substance that fills the inner cavities of bones. It is where blood is produced. Tiny spaces in the bone marrow hold blood and stem cells, the primitive cells that are able to grow into various types of blood cells. Under certain conditions, some of these stem cells can also create new tissue like bone, cartilage, fat and blood vessels. BMC, also known as bone marrow aspirate concentrate (BMAC), is a fluid containing cells taken from bone marrow.

 

HOW IS BONE MARROW CONCENTRATE (BMC) OBTAINED?

The first step in producing BMC is to collect bone marrow aspirate through a minimally invasive method that uses a needle to remove bone marrow from a large bone, such as the pelvic bone. The procedure is generally done under local anesthesia, sedation or with general anesthesia. The aspirate is then put through processing that uses a centrifuge. Bone marrow processing methods vary widely and can affect the number and quality of cells, especially in terms of useful stem and progenitor cells.

HOW IS BONE MARROW CONCENTRATE (BMC) USED TO HELP REPAIR OR HEAL TISSUE?

Stem and progenitor cells, combined with other bone marrow cells and platelets, can be used to heal tissue when injected. Furthermore, BMC contains proteins, called cytokines and growth factors, that may help heal tissue. The proposed mechanism of action is still to be determined, as well as the clinical efficacy. However, BMC is believed to decrease (modulate) inflammation and potentially aid in new tissue formation. BMC may treat bone fractures that have not grown back together, improve wound healing, help repair cartilage, treat bone death (osteonecrosis), improve tendon healing, treat osteoarthritis in joints, delay disease progression (as in arthritis)

 

BMC IN WOUND HEALING

 

Chronic wounds represent a growing healthcare burden that particularly afflicts aged, diabetic, vasculopathic, and obese patients. Normal wound healing is a complex, coordinated sequence of events orchestrated by interactions between many cell types, appropriate cytokines, and growth factors, and proceeds from hemostasis through inflammation to organized tissue regeneration.

Impaired wound healing results when these processes fail to progress through the sequential stages of healing and instead characterized by chronic inflammation and increased local injury. Apart from the obvious morbidity to the patient, these problem wounds can be a major drain on financial and human resources. Therefore, it is very important to stimulate the healing of acute and chronic wounds to a level that is not presently possible with standard care measures or recently developed innovative approaches.

Recent evidence indicates that stem cells derived from bone marrow (BM) have the potential to treat many disorders given their plasticity and ability to differentiate into various types of tissues, including skin cells. Stem cells are known to participate in cell migration, cell proliferation, and repair mechanism and rejuvenate or rebuild tissue compartments of injured tissue, as observed in chronic wounds. The BM is an important source of hematopoietic stem cells that regularly regenerate components of the blood and nonhematopoietic stem cells, including mesenchymal stem cells (MSCs). There are several potential mechanisms by which autologous stem cells could significantly contribute to wound healing. Recently, increasing focus is being placed on the use of BM-derived MSC. Previous reports also suggested that such autologous cultured cells could bring about closure of long-standing and hard-to-heal wounds.

However, even with most current therapies, >50% of chronic wounds remain refractory to treatment. Therefore, it is very challenging to identify cost-effective actual application of stem cells to reduce the frequency of nonhealing wounds. To achieve these goals, our study not only encompasses the effect of topically applied BM aspirate on chronic wounds but also provides the effect of only saline dressing in chronic wounds.

 

REFERENCES:
1.  Badis D, Omar B (2018) The effectiveness of platelet-rich plasma on the skin wound healing process: A comparative experimental study in sheep, Veterinary World, 11(6): 800-808.
2. Stem Cells in Wound Healing: The Future of Regenerative Medicine? A Mini-Review Dominik Duscher a Janos Barrera a Victor W. Wong c Zeshaan N. Maan a Alexander J. Whittam a Michael Januszyk a, b Geoffrey C. Gurtner a
3. Platelet-Rich Plasma: Support for Its Use in Wound Healing Kathleen M. Lacci, MMSc, PA-C, and Alan Dardik, MD, PhD*
4. Platelet-rich plasma: a biomimetic approach to enhancement of surgical wound healing Joseph S. Fernandez-Moure, MD, MS,a,b, * ,1 Jeffrey L. Van Eps, MD,a,b,1 Fernando J. Cabrera, MD,b Zonia Barbosa, MD,a Guillermo Medrano del Rosal, MD,b Bradley K. Weiner, MD,b,c Warren A. Ellsworth IV, MD,d and Ennio Tasciotti, PhDb
5. American Orthopaedic Foot & Ankle Society. Bone Marrow Aspiration. Accessed 3/19/2019.
6. Piuzzi NS, Mantripragada VP, Sumski A, Selvam S, Boehm C, Muschler GF. Bone Marrow-Derived Cellular Therapies in Orthopaedics: Part I: Recommendations for Bone Marrow Aspiration Technique and Safety. JBJS Rev. 2018 Nov;6(11):e4. doi: 10.2106/JBJS.RVW.18.00007. Accessed on 3/19/2019.
7. Platelet-rich plasma for the treatment of chronic wounds: evidence to date, Conde-Montero E, de la Cueva Dobao P, Martínez González JM
8. Platelet Rich Plasma: New Insights for Cutaneous Wound Healing Management Deborah Chicharro-Alcántara, Mónica Rubio-Zaragoza, Elena Damiá-Giménez, José M. Carrillo-Poveda, Belén Cuervo-Serrato, Pau Peláez-Gorrea,  and Joaquín J. Sopena-Juncosa

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