An Innovative, Centrifugation-free Method to Prepare Human Platelet Mediator Concentrates Showing Activities Comparable to Platelet-rich Plasma

Abstract: Slow-healing wounds benefit considerably from treatment with platelet-rich plasma (PrP). The drawback of using PrP is its laborious preparation, which requires expensive technical equipment (centrifuges) and well-trained personnel. Methods. The authors’ new method overcomes these issues and provides the practitioner with an innovative tool to freshly prepare a platelet mediator mix with PrP’s known biological activities, but is much simpler to obtain. This is achieved by employing the sedimentation of a blood sample at regular gravity (no centrifuge necessary) in the presence of an anti-coagulant and a sedimentation accelerator. Thereafter, the supernatant containing the platelets is concentrated on a unique filter, which causes these platelets to release their mediators (different biologically active molecules resembling the substance mix that is released by the platelets upon degranulation). This solution is eluted from the filter, providing a sterile-filtered, enriched fraction of biologically active mediators (TGF-β, PDGF, IGF-1, etc.), most of which are active in wound healing disorders. Results. This preparation triggers in-vitro proliferation of fibroblasts and osteoblasts, the secretion of IL-6 by osteoblasts, and differentiation of fibroblasts into cells with an endothelial morphology resembling cells during angiogenesis. Conclusion. By providing the practioner a sterile concentrate of a whole range of autologous platelet mediators within 1 hour, this new method has the potency to become a substitute of PrP in wound-healing therapy. PMC (platelet mediator concentrate) eases the manufacturing of such preparations, thereby making them not only more widely applicable, but also reducing treatment costs.   Accelerating the healing of chronic wounds is a major goal in human medicine. Major advances have been made in the last 20 years, most of which deal with the development of new types of wound dressing materials.¹   Approaches based on the insights modern molecular biology have also provided insights regarding cell-cell communication between the different factors collaborating to correct tissue damage (local tissue cells and the immune system). The introduction of individual recombinant human cytokines, such as PDGF, is a more recent addition to the spectrum of wound healing therapy.^2,3 Although the concept of employing genetically engineered preparations is appealing, especially considering their highly defined nature (manufactured under GMP, standardized biological activity, reproducible lot-to-lot quality), the use of either one of these cytokines alone generated disappointing results compared to mixtures of these.⁴ The latter finding can easily be explained by findings of groups that employed broader genomic or proteomic analyses to elucidate the complexity of the inter-cellular regulatory network that is established during wound healing.⁵ Therefore, it is evident that neither chronic healing is caused by the lack of a single cytokine, nor will a successful therapy be possible by applying just one of those cytokines, which are central to appropriate wound healing. Yet, an even greater obstacle to broader use of recombinant human mediators in wound healing is the dramatic increase in cost of treatment that usually results.⁶   One of the first attempts to supply such chronic wounds with more complex, physiological preparations was based on the preparation a blood platelet concentrate, usually by differential centrifugation, now known as platelet-rich plasma (PrP). Meanwhile, PrP is frequently and successfully used to improve wound healing in different areas.^7,8 One of the major advantages of PrP is that its source is usually the patient, and its active ingredients can be obtained on site. Thus, there is no need for storage and/or shipping of the platelets, which otherwise could lead to unpredictable loss in biological activity. Moreover, the use of autologous products like PrP can be regarded as safe concerning the potential presence of health risks to the patient, such as viruses or contents to which individuals may become sensitized. Certainly, a drawback of PrP is that the preparation is laborious relying not only on technical equipment, such as mid-sized centrifuges, but also well-trained personnel. This limits its use, especially by practitioners who commonly do not own such equipment, but the foremost group of specialized therapists who use such products must be regarded. In addition, it is important to note that the use of blood and blood products is subject to country-specific statutory regulations, which must be considered before using products like the ATR system.   Most patients seeking treatment prefer to visit their local practitioner opposed to a wound-healing specialist, who is typically located farther away. Therefore, it would heighten the patients’ level of comfort tremendously if they could receive such treatment where they live or even at home.

Methods

  PMC preparation. Platelet mediator concentrate (PMC) was prepared from whole peripheral blood from healthy donors using a special device ([ATR®], Advanced Tissue Regeneration, Curasan AG, Kleinostheim, Germany). All study participants gave informed consent to participate in the study. The PMC preparation consisted of 8 mL of venous blood is drawn into a 10-mL syringe, where it is mixed with a proprietary combination of an anti-coagulant and a sedimentation accelerator (Solution A and B). This blood mix is then sedimented at 1 g (desk top) for 50 mins–60 mins by placing it front end up in a side slot of the ATR system (Figure 1). A second 5 mL-syringe (“transfer syringe”) is then connected via an adaptor (front-to-front) to the blood syringe. The body of the syringe that contains the blood is then pushed down into the kit slot, which delivers the supernatant into the transfer syringe. Subsequently, 3.5 mL of this supernatant (containing the platelets) is injected into the top Luer inlet of the ATR system, where the platelets get concentrated on a filter membrane. The filter is then flushed with 2 mL of a proprietary washing buffer (Solution C) in order to eliminate any anti-coagulants and the sedimentation accelerator. Finally, injecting 0.5 mL–1.0 mL of Solution D triggers the release of mediators from the platelets (Figure 2). This is incubated at room temperature for 10 mins, after which another 0.5 mL–1.0 mL of Solution D is pressed onto the filter, thereby eluting the PMC from the filter. The PMC can be collected with a 2-mL syringe in a sterile fashion at the lateral Luer outlet. This syringe can be used to either store the PMC for a short period of time or to transfer the mediators directly onto the wound or tissue to be treated.   Unless stated otherwise, PMC was used in the different assays immediately after preparation. PMC was applied to most of the cell cultures in a final dilution of 1:10, 1:30, 1:90, and 1:270.   To study the robustness of its biological activity, PMC was also investigated using variations of the standard preparation conditions as they may occur during daily use. First, the sedimentation time was increased from 1 hour to 2 and then to 3 hours. Also, the time allowed for the release of mediators from the platelets was altered from 10 to 20 or to 40 minutes. In order to evaluate the stability of the PMC, the PMC was used in the biological assays either immediately or after 2 hours or 4 hours of storage at ambient temperature or at 4˚C–8˚C. Furthermore, PMC samples were subjected to a quick freeze-thaw process (-20˚C for 2 hours) or they were stored frozen for 2 weeks before they were tested regarding their proliferation-inducing activities in fibroblast cultures.   Cell lines. The mouse fibroblast cell line NIH-3T3 was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Cells were cultured in Dulbecco’s modified minimal essential medium ([DMEM], Biochrom AG, Berlin, Germany) supplemented with 10% fetal calf serum ([FCS], Biochrom AG), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, Deisenhofen, Germany).   The human osteoblast cell line CAL-72 was purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK). These cells were cultured in DMEM/F12 medium (Biochrom AG, Berlin, Germany) supplemented with 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 20 mM L-glutamin, and 1% insulin-transferrin-sodiumselenite (Sigma-Aldrich, Berlin). The human erythroleukemic cell line TF-1 was purchased from DSMZ. Cells were cultured in RPMI 1640 supplemented with 20% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, and 20 mM L-glutamin, as well as 20% conditioned medium derived from the human bladder carcinoma cell line 5637 (also purchased from DSMZ).   Analysis of biological activities of PMC. For the analysis of the biological activity of PMC, the human osteoblast cell line CAL-72 was cultured at a density of 2500 cells per well in 96 well microtiter plates overnight in medium containing 10% FCS. Then the cells were starved for the next 24 hours by reducing the FCS concentration to 2%. This was followed by the addition of PMC at different concentrations or of 10% of FCS (positive control) for another 48 hours. Cells were washed once and the vital dye acridine orange (1 µg/mL, Sigma) was added. After washing the cells, intra-cellular fluorescence was measured in a 96-well plate reader (POLARstar, BMG Labtech, Offenburg, Germany). The supernatants of the same experiment were analyzed for the release of IL-6 into the culture medium (by sandwich ELISA using the IL-6 matched antibody pair, [R&D Systems GmbH, Wiesbaden, Germany]). Analyses were performed in triplicates.   NIH-3T3 cells were seeded at a density of 2000 cells per well into a 96 well microtiter plate and allowed to adhere overnight in complete medium followed by 4 hours of starvation. Afterward, PMC was added at different concentrations in triplicates for 48 hours. For the different tests, either freshly prepared PMC was used or PMC stored for different conditions and periods (see above). Fetal calf serum at 10% served as positive control. Microscopical analysis was performed using an inverted microscope ID 03 (Carl Zeiss Jena GmbH, Jena, Germany). Proliferation of NIH-3T3 cells was assessed using the MTS test. Briefly, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) was added to the cells for 4 hours using the “cell titer 96 aqueous one solution cell proliferation assay” (Promega GmbH, Mannheim, Germany). The test was performed strictly according to the manufacturer’s instructions.   TF-1 cells were seeded at a density of 5000 cells per well and allowed to adhere overnight followed by 4 hours of serum deprivation. Thereafter, cells were cultured in the presence (positive control) or absence (neg. control and PMC) of the conditioned supernatant of 5637 cells. PMC dilutions were added in triplicates for 24, 48, and 72 hours. The proliferation of TF-1 cells was measured using the MTS test as described above.   Analysis of mediator composition of PMC and mediator release of CAL-72. Interleukin-6 (IL-6), transforming growth factor beta (TGF-β) and platelet-derived growth factor-AB (PDGF-AB) were detected in PMC, as well as in the supernatant of CAL-72 cells, by sandwich ELISA (using matched pair antibodies and standard proteins from R&D Systems GmbH).   Other mediators present in PMC were measured by using the multiplexed analyte profiling (MAP) technology (Rules-Based Medicine Inc., Austin, TX), which was done for brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), basic fibroblast growth factor (FGFb), growth-related oncogene-alpha (GROa), stem cell factor (SCF), as well as vascular endothelial growth factor (VEGF).

Results

  Mediator composition PMC. Platelets are the source of various types of mediators critical for the regulation of hemostasis and wound healing. The preparations of three different healthy volunteers were examined using a multiplexed assay measuring 8 different cytokines and chemokines in an attempt to characterize the composition of PMCs. In all samples it was shown that the concentrations in the PMCs were substantially higher as in the plasma samples obtained from the same donors together with the blood samples from which the PMC was prepared (Table 1).   Validation of the preparation of PMC. One of the most important quality criterion for biological systems is to show their variability, which was achieved by preparing 6 PMCs from two different blood donors. The intra-donor coefficients of variation (CV%) were found to range below 12%, which can be considered satisfactory for a primary cell-based system (Table 2).   It is of equal importance for such biological samples to assess the range of cytokine concentrations that will have to be expected in PMCs manufactured from the blood of different donors. It is well known that inter-individual differences, especially with regard to cytokine concentrations in freshly prepared primary cells or in cultures hereof, are usually much higher than the variations seen with repeated preparations of the same donor. The manufacturing of the PMCs obtained from the blood of 6 different healthy donors gave a CV of 26.4% (Table 3).   Biological activities of PMC. Aside from the molecular characterization of PMC, it was important to characterize its bioregulatory effects relevant to wound healing. Compared to tissues in vivo, cell cultures in microculture trays differ in one important aspect: in vivo the wound communicates with the neighboring area via the constant diffusion process between the local tissue fluid and the interstitial fluid of surrounding tissues on the one hand, the blood and lymph vessels on the other. Thus, diffusion will dilute all drugs applied to the wound, which will form an efflux gradient away from the wound; this is not the case in vitro. Whatever is tested in cell cultures will be available over the entire incubation period at approximately its starting concentration. Considering this, the authors chose a 1:10 dilution of the concentrated mediator mix as highest final PMC concentration in the cultures. The serial dilution to complete the dose-response curves were 1:30, 1:90, and 1:270 (all final concentrations).   Activities of PMC on fibroblasts. Fibroblasts are one major target for mediators released by platelets in wounds. Therefore, the PMC was first tested in fibroblast cultures where a clear dose-dependent increase of the proliferation of these cells was found (Figure 3). In this culture a supra-optimal concentration was identified, which mediated less cell growth support than lower concentrations. This supra-optimal effect could not be detected in proliferation experiments using osteoblasts, where also an increase in cell proliferation was observed (Figure 4), nor with a leukemic cell line (Figure 5). Thus, this supra-optimal concentration range of the platelet mediators seems to be cell-type specific for these fibroblasts.   PMC activities in osteoblast cultures. Another cell type important for the healing of wounds, in which the bone is damaged, is the osteoblast. CAL-72, an osteoblast cell line, was therefore employed to examine, which effects PMC has on these cells. Also in this case the proliferation was chosen as one of two different endpoints, because in most cases bone regeneration following injury requires a propagation of local osteoblasts. The second parameter was the synthesis and release of IL-6, a pluripotent cytokine secreted during osteoblast activation. Both of these parameters were found to be stimulated by PMC (Figure 4).   Lack of supporting the proliferation of leukemic cells. Increasing the proliferation of cells can bear the risk of promoting the growth of leukemic cells. Although this risk is rather low, especially regarding the fact that the preparation contains nothing, but constituents released from autologous platelets, it was important to also test a leukemic cell line (TF-1, an erythroleukemic cell) to see whether there is also growth support with such cells.   The proliferation rate of TF-1 cells was unchanged in these experiments (Figure 5).   Robustness of the preparation of PMC. The onsite preparation of PMCs in physicians’ practices will interfere with stressful schedules. This may lead to deviations from the protocol as described in the instruction manual. One of the major goals of this development process was to examine a reasonable range of variability for the single steps for the preparation of PMCs. This comprised three different aspects:     a) the time the blood is allowed to sediment     b) the time the release takes     c) the period of storing the readily prepared PMCs before use.   The results of these investigations are presented in Figures 6–9.   The tests for robustness demonstrated a considerable tolerance of the method against errors as they can occur in practices or hospitals. Moreover, it was shown that the mediator concentrate could be stored frozen, which provides the therapist with the opportunity to prepare a larger batch and freeze aliquots for later use.   One of the findings in these experiments was that at a final dilution of 1:10 of the original PMC in the cultures (highest concentration tested) the proliferation of the fibroblast target cells was less pronounced than at the 1:30 dilution (“supra-optimal” effect).   Other biological activities of PMC in vitro. In addition to supporting the proliferation of fibroblasts, PMC also induced morphological changes in these cells. While the addition of the positive control (fetal calf serum) generated the picture of a homogenous confluent cell layer, the PMC cytokine mix lead to a morphological change in the growth pattern of fibroblasts, which was obviously accompanied by a reduced proliferation rate. This newly acquired morphology of the fibroblast cultures, which occurred with the PMCs of all three donors tested so far, reminded of growth of endothelial cells starting to form tubules in vitro (Figure 10).

Discussion

  The results shown above demonstrate that the centrifugation-free PMC preparation yields substantial quantities of both of the most important mediators for wound healing, TGF-β and PDGF. Moreover, when performing a multiplexed mediator analyses, PMC also proved to contain other important components of platelet granules, which can all be considered important to wound healing, such as BDNF,⁹ EGF,¹⁰ FGF-β,¹¹ GRO-α,¹² SCF,¹³ as well as VEGF.¹⁴   The concentration of these mediators in PMC was considerably higher than those found in the plasma of the same donors: BDNF was increased by an average factor of more than 300, PDGF by a factor of ca. 100, and despite the fact that TGF-β is always present at considerable concentrations in plasma,¹⁵ this growth factor was also concentrated more than 50-fold. Another major advantage of PMC is that it not only can be prepared freshly from each patient to treat wounds with autologous cytokines. The latter not only precludes the introduction of foreign antigens into open wounds (eg, blood group antigens, HLA molecules), but also altered protein structures from many recombinant human cytokines and other proteins leading to immunogenic responses.¹⁶   The biological activity of PMC was shown using two of the most important cell types during wound healing: fibroblasts (for superficial and other wounds) and osteoblasts (for deeper wounds and/or bone defects). The proliferation of both, which our experiments demonstrated to be induced by PMC, is considered an important feature in wound healing.^17,18   In experiments showing an enhancement of fibroblast proliferation a supraoptimal effect was observed at the highest concentration of PMC, causing a more or less pronounced reduction of the maximal PMC effect. This is a well-known phenomenon, especially in fibroblasts, but also other cells, and was described for various protein mediators, such as basic FGF,¹⁹ EGF,²⁰ as well as TGF-β^21–23 or PDGF.²⁴ Moreover, the same type of bell-shaped dose-response relationship was even found with platelet-rich plasma.^25,26 Hence, this can be regarded as a general physiological mechanism, the real meaning of which to wound healing and tissue turnover still waits to be elucidated.   The dose-response relationship that was found for PMC shows that it dose-dependently increases the activities of the osteoblasts tested, without any supraoptimal concentration range detectable. It is highly likely that the decrease of PMC activity seen at higher concentrations in the fibroblast cultures more than likely will not be relevant in vivo. As previously mentioned (see Results), the wound fluid communicates with adjacent compartments (interstitial fluid, blood, lymph of the surrounding tissue) by diffusion. Concerning any given substance applied to the wound, this process generates an efflux over time out of the wound and into the adjacent tissues, reducing the local concentration and forming a concentration gradient. Therefore, those concentrations of the PMC mediators shown to support fibroblast growth and angiogenesis-like effects optimally can in fact be expected to occur within the tissue forming the wound edges, rather than in the wound surface itself. This is important because the particular cells located in the rim area of wound initiate the healing process.   On the other hand, PMC does not support the growth of leukemic cells. This not only demonstrates the cell-type specificity of its effects, but can also be regarded a safety indicator of PMC.   Another interesting activity of PMC was seen upon microscopic inspection of those cultures used to investigate fibroblast proliferation. The cells incubated with PMC in these cultures were even found to develop morphological features of angiogenesis. The general property of fibroblasts to turn into an angiogenic phenotype was also observed by others.^27,28 Such fibroblast activities can be expected in vivo to improve conditions, especially in chronic wounds where an insufficient blood supply has been identified as an important factor of delayed healing. An accelerated angiogenesis was also correlated to improved healing rates by others.²⁹ Pro-angiogenic activities in PMC would surely serve as another valuable building block of its therapeutic activities.   Whenever wounds are accompanied by bone injury, osteoblasts are required to contribute to the healing process.¹⁸ The authors’ investigations showed that PMC was not only able to increase the replication rate in osteoblast cultures, but also to cause these cells to secrete more IL-6 simultaneously. The latter cytokine is known as one of the major factors, contributing in various ways to bone turnover.³⁰   Eventually, the ease of preparing PMCs will enable the use of such preparations also outside hospitals or wound-treatment centers. Moreover, the freeze-thaw stability of the biological activity of the mediators in PMC should even allow the preparation of larger batches of autologous mediators, allowing a convenient subsequent storage of aliquots at -20˚C, which can be used for follow up treatments, not only eliminating the need for another blood draw, but also reducing substantially the workload of the practitioner, as well as the time each patient needs to wait for being treated during the follow-up visits.   Surely, the complexity of the PMC mediator mixture is one of its outstanding features. Most abundant among the mediators found in PMC is TGF-β (at least 50 ng/mL up to more than 100 ng/mL), as well as PDGF (usually ~30 ng/mL). Barrientos et al31 conducted a review of the many articles that describe the role of TGF-β and PDGF in acute and chronic wound healing. It was recognized quite early in the use of recombinant growth factors in wound healing that treating wounds by means of single mediators produced limited success compared to combinations of cytokines, (eg, TGF-ββ plus PDGF).⁴ Therefore, the authors expect PMC, consisting of a whole variety of freshly prepared chemokines and growth factors, to exhibit superior efficacy compared to single growth factor therapy. Investigations describing the highly complex patterns of genes activated in healing tissues in vivo underscore this view.⁵

Conclusion

  Compared to traditional PrP systems in tissue and bone regeneration, the authors expect improved results with the use of PMC, as a result of the high concentrations of growth factors and their “pro-angiogenic” effects. Gaining more PMC with the ideal dilution and the option of storing frozen PMC will make this product even more interesting under economic perspectives.

Acknowledgements

  This development was sponsored by Curasan AG (Kleinostheim, Germany). Dr. Schmolz discloses that he has received compensation from Curasan AG for contract development. Dr. Stein discloses that he has received speaker honoraria from Curasan AG. Dr. Hübner is an employee of Curasan AG.

References

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