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Article

Industrial Mass Production of Platelet Dry Powder

by
Shih-Hung Lin
1,
Chen-Ying Su
1,2 and
Hsu-Wei Fang
1,2,3,*
1
Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei 10608, Taiwan
2
High-Value Biomaterials Research and Commercialization Center, National Taipei University of Technology, Taipei 10608, Taiwan
3
Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli 35053, Taiwan
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(23), 12870; https://doi.org/10.3390/app132312870
Submission received: 1 November 2023 / Revised: 24 November 2023 / Accepted: 28 November 2023 / Published: 30 November 2023
Browse Figures
Versions Notes

Abstract

:
The goal of this paper is to examine the use of pig blood in the industrial mass production of platelet dry powder and to transform platelet dry powder into a low-cost and mass-produced material. Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) contain multiple types of growth factors (GFs) and can be widely used in medical applications. However, neither can be mass-produced, due to the complexity of the PRP preparation process and the lack of anticoagulants in the PRF preparation process, increasing the risk of coagulation during mass production. Another obstacle is the insufficient supply of autologous PRP and autologous PRF. In this study, platelet dry powder was mass-produced from pig blood through the indirect addition of calcium chloride solution. Furthermore, the results showed that different concentrations and percentages of calcium chloride solution had significant effects on concentrations of TGF-β1 and PDGF-BB in the platelet dry powder. The platelet dry powder mass-produced from pig blood demonstrated high concentrations of GFs and long-term shelf stability, increasing the supply to industries that use it in product development.

1. Introduction

Platelets contain many types of growth factors (GFs): platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), transforming growth factor-beta (TGFβ), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), among others [1,2,3,4,5,6,7,8]. GFs are essential at every stage of physical healing, and consequently, the regenerative properties of platelets have received heightened interest and increased use in various medical fields. Common platelet derivatives include platelet-rich plasma (PRP) [1,2,3,4,5,6] and platelet-rich fibrin (PRF) [2,3,5].
PRP is prepared by mixing collected blood with a certain amount of anticoagulant and centrifuging the mixture until the blood separates into layers [9,10,11]. The uppermost layer is platelet-poor plasma (PPP), and the middle layer is PRP; the bottom layer is predominantly red blood cells. The PRP is then carefully extracted with a syringe to avoid drawing any red blood cells or PPP.
PRF is produced from small blood samples and without the addition of any anticoagulants during the preparation process [12,13]; the blood sample is centrifuged immediately after being drawn and will begin to coagulate during the process, dividing into three layers inside the vial: red blood cells in the bottom layer, plasma-poor platelets in the supernatant, and PRF clots in the middle. PRF clots are rich in fibrin, platelets, and leukocytes.
When preparing PRP, to ensure the highest concentration of platelets, the syringe must be carefully wielded to avoid the red blood cells and PPP while extracting the PRP layer. Due to this need for precision, PRP preparation is a time-consuming operation, and the time cost is increased when processing large amounts of blood. Time constraints are an impediment to the mass production of PRP.
Because anticoagulants are not used when preparing PRF [12,13], once collected, blood must be centrifuged immediately before it can congeal. Consequently, PRF preparation cannot be carried out on an industrial or mass scale. In particular, when processing large amounts of blood, a delay in any step may increase the risk of blood coagulation.
The inability to mass produce PRP and PRF—due to the complex process of preparing the former and the risk of coagulation in the latter—has limited the range of their applications. The use of PRP and PRF is mainly focused on autologous applications [14,15,16]. Nonetheless, due to the constraints associated with human autologous blood, scaling up to mass production presents significant challenges. The inability to produce sufficient amounts means the inability to meet large-scale product demands, as well as the inability to prepare diversified dosage forms tailored to specific applications, limiting the possible commercial applications of platelet dry powder.
Within platelets, there are mainly three types of granules: alpha granules, dense granules, and lysosomes [6,7,8], each containing different bioactive substances. When platelets are activated by the addition of activators, they release growth factors (GFs) and other bioactive molecules stored in these various granules [5,17,18,19]. During this activation process, the bioactive substances released from the granules initiate a range of biological reactions, including platelet aggregation, cell proliferation, and cell migration.
In the preparation of platelet products, activating platelets [14,20,21] is essential, and is commonly achieved by adding activators like thrombin, collagen, and calcium chloride solution [17,22]. Thrombin, often used, is typically derived from bovine sources, posing risks such as high costs and potential immune reactions or allergies in patients [6,22,23,24]. To mitigate these risks, particularly the immunological and transmission concerns related to bovine thrombin, this study utilizes calcium chloride solution as a safer activator alternative.
On the other hand, current medical settings are constantly beset by severe blood shortages, further hindering the mass-production of platelet dry powder; hence, the urgent need to replenish the blood supply with blood from alternative species. Pig blood has become an ideal option—easy to obtain and with a stable and abundant supply. More importantly, pigs are already widely used in biomedical research, and familiarity with their physiological and hematological properties allows us to adapt and maximize their utilization with ease. Therefore, the decision to use pig blood to produce platelet dry powder is not only practical, it is based on inherent biological and economic advantages.
The objective of this study was the preparation of platelet dry powder through a novel method using pig blood as a raw material that can yield a sufficient output of stable quality to meet different clinical needs. The ultimate goal is the widespread use of platelet dry powder in product development in medical fields.

2. Materials and Methods

2.1. Platelet Dry Powder Preparation

Whole pig’s blood was mixed with citrate phosphate dextrose as an anticoagulant at a 50:7 ratio to prevent agglutination of the blood. The blood was centrifuged at a low speed to separate it into layers, with most red blood cells precipitating in the lower layer of the centrifuge vial. The upper layer was plasma containing platelets. This upper layer was harvested.
Platelet dry powder was prepared through the indirect addition of an aqueous calcium chloride (10043-52-4, Merck Sigma-Aldrich, Steinheim, Germany) solution. First, a small amount of plasma containing platelets was added into a calcium chloride solution with a concentration of 0.01–0.00001 M and mixed evenly to form Mixture A. Platelet-containing plasma was added to this mixture at percentages of 1–20% and mixed thoroughly to form Mixture B, which was centrifuged at a high speed. The upper layer of liquid was removed, leaving behind platelet wet powder at the bottom of the vial. The wet powder was freeze-dried, resulting in platelet dry powder. The freeze-dried platelet powder was then sterilized with gamma radiation for subsequent testing.

2.2. Effects of Calcium Chloride Solution Concentration on Platelet Concentration

Four variations of Mixture A were created by mixing platelet-containing plasma with calcium chloride solutions of four different concentrations, which were 0.01 M, 0.001 M, 0.0001 M, and 0.00001 M. These four mixtures were then thoroughly mixed with platelet-containing plasma at a 1:9 ratio to form variations of Mixture B. Mixture B was spun in a centrifuge at high speed and separated into layers. After removing the upper liquid layer, platelet wet powder remained. A hemocytometer was used to measure platelet concentration of the whole pig blood and centrifuged platelet wet powder in order to determine whether different calcium chloride concentrations produced any changes in platelet concentration and what effects it had on platelet functions.

2.3. Effects of Calcium Chloride Solution Percentages on Platelet Concentration

A solution with a calcium chloride concentration of 0.01 M was mixed with a small amount of platelet-containing plasma to form Mixture A, which was further mixed with platelet-containing plasma at different percentages to create variations of Mixture B: 1%, 2%, 5%, 10%, and 20%. Mixture B was spun in a centrifuge at high speed and separated into layers. The upper layer of liquid was removed, leaving behind platelet wet powder at the bottom of the vial. A hemocytometer was used to measure platelet concentration of the whole pig blood and centrifuged platelet wet powder in order to determine whether different calcium chloride concentrations produced any changes in platelet concentration and what effects it had on platelet functions.

2.4. Effects of Calcium Chloride Concentrations on TGF-β1 and PDGF-BB

Four variations of Mixture A were created by mixing platelet-containing plasma with calcium chloride solutions of four different concentrations, which were 0.01 M, 0.001 M, 0.0001 M, and 0.00001 M. These four mixtures were then thoroughly mixed with platelet-containing plasma at a 1:9 ratio to form variations of Mixture B. Mixture B was spun in a centrifuge at high speed and separated into layers. After removing the upper liquid layer, the remaining platelet wet powder was freeze-dried into platelet dry powder.
The TGF-β1 and PDGF-BB contents of the platelet dry power were tested using commercially available Sandwich ELISA test kits. TGF-β1 was tested with the Human/Mouse/Rat/Porcine/Canine TGF-beta 1 Quantikine ELISA kit from R&D Systems, Catalog #DB100C. PDGF-BB was tested using the PDGF-BB Porcine ELISA kit by Thermo Fisher.

2.5. Effects of Calcium Chloride Percentages on TGF-β1 and PDGF-BB

A solution with a calcium chloride concentration of 0.01 M was mixed with a small amount of platelet-containing plasma to form Mixture A, which was further mixed with platelet-containing plasma at different percentages to create variations of Mixture B: 1%, 2%, 5%, 10%, and 20%. Mixture B was spun in a centrifuge at high speed and separated into layers. After the upper layer of liquid was removed, the remaining wet powder was freeze-dried into platelet dry powder. The TGF-β1 and PDGF-BB contents of the platelet dry power were tested to determine the release of GFs in the platelets.

2.6. Reproducibility across Different Batches

To ensure that the mass-production process is replicable and stable, different batches of platelet dry powder were prepared under the same conditions. A solution with a calcium chloride concentration of 0.01 M was mixed with a small amount of platelet-containing plasma to form Mixture A, which was further mixed thoroughly with 20% of platelet-containing plasma to create Mixture B. Mixture B was spun in a centrifuge at high speed and separated into layers. After the upper layer of liquid was removed, the remaining wet powder was freeze-dried into platelet dry powder. GF testing was performed on each batch of platelet dry powder to monitor reproducibility across different batches.

2.7. Platelet Dry Powder Storage

The prepared platelet dry powder was vacuum-sealed and stored at room temperature. GF testing was performed after 6 months and 12 months. Stability and activity of platelet dry power was evaluated according to the storage and loss of GF in such conditions in order to ensure that the platelet dry powder can be reliably used in future studies and applications.

2.8. Statistical Analysis

The results of experiments 2.6 and 2.7 were based on six independent experiments. The results of experiments 2.1 to 2.5 were derived from three independent experiments and expressed as the averages ± standard deviations. In the paired sample t-tests, p < 0.05 is considered as significant difference.

3. Results

3.1. Effects of Calcium Chloride Solution Concentration on Platelet Concentration

The different concentrations of calcium chloride solution were 0.01 M, 0.001 M, 0.0001 M, and 0.00001 M. The experiment results show that, regardless of the concentration, the platelet concentration never dropped below 3000 × 103/μL; compared with the platelet concentration of whole blood, the addition of different concentrations of calcium chloride solution resulted in platelet wet powder with 15 times the platelet concentration of whole blood, with no significant differences in platelet concentration across the calcium chloride concentrations.
Figure 1 depicts the platelet concentration of platelet dry powder prepared with solutions with calcium chloride concentrations of 0.01 M, 0.001 M, 0.0001 M, and 0.00001 M. Platelet concentration across these four concentrations of calcium chloride were all in excess of 3000 × 103/μL, 15 times the platelet concentration of whole blood. However, the differences in platelet concentration across the platelet wet powder was not significant (average ± SD, n = 3).

3.2. Effects of Calcium Chloride Solution Percentages on Platelet Concentration

The different percentages of calcium chloride solution were 1%, 2%, 5%, 10%, and 20%. The experiment results show that, regardless of the solution percentage, the platelet concentration never dropped below 3000 × 103/μL; compared with the platelet concentration of whole blood, the addition of different percentages of calcium chloride solution resulted in platelet wet powder with 15 times the platelet concentration of whole blood, with no significant differences in platelet concentration across different percentages of calcium chloride solution.
Figure 2 depicts the platelet concentration of platelet dry powder prepared with 1%, 2%, 5%, 10%, and 20% calcium chloride solution. Platelet concentration across these five percentages of calcium chloride solution were all in excess of 3000 × 103/μL, 15 times the platelet concentration of whole blood. However, the differences in platelet concentration across the platelet wet powder was not significant (average ± SD, n = 3).

3.3. Effects of Calcium Chloride Concentrations on GFs

Figure 3a demonstrates that, regardless of the concentration of calcium chloride, the resulting TGF-β1 concentrations were 80,000 pg/mL or higher; furthermore, the sample groups with 0.01 M and 0.0001 M concentrations had significantly higher concentrations of TGF-β1 than the other samples, exceeding 100,000 pg/mL. As seen in Figure 3b, all concentrations of calcium chloride resulted in PDGF-BB concentrations in excess of 60,000 pg/mL and as high as 88,000 pg/mL with the addition of 0.001 M concentration calcium chloride, significantly higher than the other sample groups. Different activators affect different GFs [17], and the experiment results also show that adding different concentrations of calcium chloride solution will produce different effects on GFs. Consequently, in practical applications, the concentration of calcium chloride can be selected to activate the necessary GFs according to the specific requirements and field of application.

3.4. Effects of Calcium Chloride Percentages on GFs

The experiment results show that, regardless of the solution percentage, the resulting TGF-β1 concentrations were 80,000 pg/mL or higher; furthermore, the sample group with 20% calcium chloride solution had a significantly higher concentration of TGF-β1 than the other groups, exceeding 100,000 pg/mL. As seen in Figure 4b, all concentrations of calcium chloride resulted in PDGF-BB concentrations in excess of 70,000 pg/mL; furthermore, PDGF-BB concentration was highest in the sample group with 10% calcium chloride solution, exceeding 90,000 pg/mL. The figures also reveal that, while the preparations with 1%, 2%, and 5% calcium chloride solutions did not yield the highest TGF-β1 or PDGF-BB concentrations, the outcomes were still promising. However, from a mass-production perspective, 5% to 19% more blood can be processed in the same amount of time with the addition of 1%, 2%, or 5% calcium chloride solution, compared to the addition of 20% calcium chloride solution. Therefore, these three lower percentages may be more desirable if the consideration is to increase production capacity.

3.5. Reproducibility across Different Batches

Six independent batches of platelet dry powder were produced with 20% calcium chloride solution with a concentration of 0.01 M in order to test their TGF-β1 and PDGF-BB concentrations. As can be seen in Figure 5a,b, the TGF-β1 concentrations were in the 120,000–140,000 pg/mL range, with a mean concentration of 129,080 pg/mL, and the PDGF-BB concentrations were in the 70,000–90,000 pg/mL range, with a mean of 81,173 pg/mL. The experiment results indicate high reproducibility across these six batches of dry powder, with little variation between batches, demonstrating the stability and repeatability of the preparation process. The results further reveal that, although TGF-β1 and PDGF-BB concentrations are affected by several factors, including the centrifugation conditions during preparation, the activation process, and the freeze-drying conditions, the variations between platelet dry powder batches were minimized and consistency between batches was ensured through a series of rigorous experiment operations and optimized preparation conditions. Furthermore, the stable release of GFs further demonstrates the repeatability and reliability of the preparation process.

3.6. Shelf-Life Testing

Six batches of platelet dry powder were prepared and vacuum-sealed for storage, and the TGF-β1 concentrations were tested when preparation was completed and both 6 and 12 months after completion. Figure 6 shows that at least 90% of the initial TGF-β1 concentration was still present after 12 months, demonstrating the long-term stability and reliability of the platelet dry powder, while also verifying the stability of the preparation process and the repeatability of the experiment.
Figure 6 depicts the TGF-β1 concentrations of six batches of platelet dry powder prepared under the same conditions, then vacuum-sealed and stored at room temperature. After 12 months, each batch of platelet dry powder retained at least 90% of their initial TGF-β1 concentrations. These results demonstrate the long-term stability and reliability of the platelet dry powder, while also verifying the stability of the preparation process and the repeatability of the experiment.

4. Discussion

Industrial mass-production of platelet dry powder was performed with pig blood. The centrifuge used in this study can process over 300 L of pig blood per hour, yielding large amounts of wet powder that was freeze-dried into platelet dry powder. The experimental results indicate that the addition of calcium chloride solutions at different concentrations did not result in significant differences in platelet concentrations among the respective concentrations. Similarly, when different percentages of calcium chloride solutions were added, no noticeable variations in platelet concentrations were observed among the respective percentages. These findings suggest that, under the conditions of these experiments, the concentration and percentages of calcium chloride do not exert a discernible influence on platelet quantity. However, in contrast to platelet concentration, the results clearly demonstrate a pronounced impact of different concentrations and percentages of calcium chloride solutions on TGF-β1 and PDGF-BB concentrations. This highlights the significance of calcium chloride in influencing the release of growth factors, potentially impacting subsequent applications. Therefore, when utilizing calcium chloride solutions, careful regulation of TGF-β1 and PDGF-BB is crucial, in addition to monitoring platelet concentration, to ensure the accuracy and effectiveness of platelet dry powder applications. GF testing of the sterilized platelet dry power showed TGF-β1 concentrations over 100,000 pg/mL and PDGF-BB concentrations over 80,000 pg/mL, demonstrating that our method of preparing platelet dry powder is both high yield and high quality. Furthermore, our proposed method circumvents the risk of coagulated blood in the preparation of PRF without the use of anticoagulants [12,13] and the need for the careful extraction of platelets in the preparation of PRP [9,10,11], saving time and preventing human error. The two GFs tested in this study were TGF-β1 and PDGF-BB; however, the lesser availability of GF detection reagents for pig models than for human or mouse models has limited our comprehension and evaluation of the various GF contents in platelet dry powder prepared with pig blood to some extent.
During the preparation of platelet products, the activation of platelets [14,20,21] is a vital step. There are many ways of activating platelets, including the addition of activators, the cyclical freezing and thawing of platelets, and the use of ultrasound techniques [22]. The addition of activators is broadly considered to be a more efficient method of activation, and common activators include thrombin, collagen, and calcium chloride solution [17,22]. As the platelet dry powder can be prepared in various dosage forms for human use, when selecting an activator, its associated risks must also be considered. Thrombin may be a common option, but most thrombin products are derived from bovine thrombin, which is costly and holds certain risks that cannot be ignored. The use of exogenous bovine thrombin may trigger immune responses and allergic reactions in patients [6,22,23,24]. The use of thrombin from either homologous or xenogenic sources in this procedure carries substantial risks. These forms of thrombin, derived from either genetically similar or distinct species, have the potential to induce allergic reactions in patients. The natural sensitivity of the human immune system to unfamiliar proteins, especially those identified as foreign, may lead to negative immunological reactions upon contact with these forms of thrombin. Moreover, the use of xenoproteins, particularly those originating from animals, presents significant concerns regarding the transmission of pathogens. These infectious agents, which include a range of viruses, bacteria, and other microorganisms, might be harmless in their original hosts but can become serious health threats when transferred to humans. This risk is even more acute in immunocompromised patients, who are more susceptible to infections. Therefore, it is critical to carefully consider the use of thrombin in the activation of PRP, and to implement strict safety measures to minimize these health risks. Therefore, calcium chloride solution was selected as the activator in this study to avoid the potential immunity and transmission risks associated with bovine thrombin.
In the activation process of PRP, meticulous dosage control is imperative, regardless of whether thrombin or calcium chloride is employed as the activating agent. Excessive use of these activators can interfere with the normal coagulation mechanisms [25,26,27], posing additional risks, particularly for patients who are on anticoagulant therapy or have coagulation disorders. Furthermore, over-activation may lead to the premature release of growth factors from platelets, adversely affecting the therapeutic efficacy of PRP. Hence, precise dosing and consideration of the patient’s specific medical condition are crucial in the administration of these substances in PRP therapy to ensure both the safety and effectiveness of the treatment.
The effects of calcium chloride solution in various concentrations and percentages on the activation of GF has been examined in this study. The literature on this topic also mentions three types of activators—collagen, thrombin, and calcium chloride—and their effects on GF release when used on their own or as a mixture. Different activation combinations will produce significant differences in the performances of various GFs such as TGF, PDGF, IGF, KGF, and VEGF [5,17,28,29], and the activation effect of thrombin or a mix of these three activators is generally superior to that of calcium chloride alone [17,28,29]. Furthermore, when high concentrations of calcium and thrombin are used, they will quickly induce significant increases in TGF-β1 and PDGF concentrations, which will remain essentially constant for 6 days; in contrast, lower concentrations may reduce and delay the release of GFs [29]. This demonstrates that different combinations of activators will have different effects on the length of GF release times. The combination of different activators significantly affects the amount and duration of GF release. The selection of a specific combination of activators depends not only on the type of GF required but also on the desired timeframe for GF release. For instance, in clinical applications where a rapid and substantial release of a specific GF is necessary, a combination of activators that can quickly facilitate GF release may be chosen. Conversely, in scenarios requiring a slow and sustained release of GF to support long-term tissue repair or regeneration, a combination that allows for a gradual release of GF might be preferred. The choice of activators in different medical applications requires critical guidance, especially in the fields of regenerative medicine and tissue engineering, where the pattern of GF release is crucial for therapeutic effectiveness. Therefore, a deeper understanding of the mechanisms by which different activator combinations affect GF release will aid in optimizing the clinical application of products, thereby enhancing the efficiency and efficacy of treatments. In mass-production situations, the choice of activator is not solely based on the activation of different GFs, but also requires consideration of other factors such as cost. Calcium chloride is generally lower cost than collagen and thrombin, which are quite expensive. Different requirements will also determine which activator is chosen. For example, activators that can rapidly release TGF-β1 in large amounts may be prioritized in applications to promote bone repair, while activators that assist with the steady release of PDGF and KGF may be preferred in hair follicle regeneration. Furthermore, the dosage form may also influence the activator choice, as the efficacy and adaptability of the activator must also be considered when creating a gel dosage form used to dress wounds or liquid dosage form to spray on skin. Calcium chloride solution was selected as the activator in this study because of its lower cost and ability to induce the formation of natural thrombin, thus simulating the physiological process and the sustained release of GFs. Sustained GF release is critical to ensuring proper tissue repair and wound healing. However, in the future, different activator combinations may be considered according to the GFs and applications involved.
In common PRP applications, the activation of platelets [12,14,15,17,20,24] is seen as a vital step in the release of GFs, which play a key role in cell proliferation, migration, and differentiation. The activation process is dependent on the use of specific activators, which are commonly thrombin, collagen, and calcium chloride solution [5,6,17]. Platelets contain three primary granule types: α granules, dense granules, and lysosomes [6,7,8]; each granule stores different bioactive substances. When platelets are activated through the direct addition of these activators, GFs and other bioactive substances stored in these granules are released [5,17,18,19]. During the activation process, these granules will release their bioactive substances and thus induce biological responses such as platelet aggregation, proliferation, and cell migration. The direct addition of an activator to activate PRP will alter the structure of fibrinogen into a 3D fibrin structure, converting fibrinogen into fibrin gel [5,8,17,18]. This gel structure not only acts as a GF carrier, it also provides a microenvironment with controlled release properties, allowing the GF to be released when and where needed. However, in the mass-production of PRP, ensuring that all the completed PRP has been transformed into fibrin gel is incredibly difficult and time-intensive; furthermore, partial transformation of the platelet solution into fibrin gel will compromise the concentration and quality of the resulting platelet dry powder. Therefore, preventing the formation of fibrin gel is crucial in mass-production processes. In this study, platelet activation through the indirect addition of calcium chloride solution both enhanced the release of GF and minimized the formation of fibrin gel, ensuring that the concentration and quality of the prepared platelet dry powder was up to standard.
In regenerative medicine research, PRP and PRF applications are primarily centered around autologous use [14,15,16]. However, given the limitations of human autologous blood, large-scale mass-production is rife with difficulties. In contrast, pig blood as the source for preparing platelet dry powder guarantees a steady supply that is easy to obtain and relatively low-cost, making it the foremost choice for the mass-production of platelet dry powder. Moreover, given the widespread use of pigs in biomedical research, our profound understanding of their physiological and hematological properties provides a solid foundation for their use in regenerative medicine. Studies have shown favorable effects among animal models when using PRP from different species in allogeneic or heterogeneous treatments; examples include the use of canine PRP gel on wound healing among rabbits [30], the use of canine PRP on treating skin wounds among cats [31], the extraction of GFs from equine blood on repairing cartilage defects among rabbits [18], the use of equine PRP treatments to accelerate healing in rabbits with surgically induced skin wounds [32], and human PRP to induce apoptosis in rabbit chondrocytes [33]. These examples demonstrate the applicability of platelets and GFs between species. Successful examples of cross-species applications of platelets and GFs allow us to find versatile and effective platelet and GF applications through in-depth research, contributing to the further development of regenerative medicine. In addition to further research into the role of GFs in autologous repair and regeneration, a new research direction will be their cross-species applications and effects.
Numerous studies have reported the application of freeze-dried PRP or PRF in areas such as wound healing [34], bone repair [18], and ophthalmic diseases [35]. Compared to fresh autologous PRP, the shelf-life of freeze-dried growth factors is significantly longer, typically ranging from 12 to 18 months [18], as opposed to only 4 to 8 h or even less for autologous PRP. Recent research has confirmed that freeze-dried growth factors significantly enhance the healing of induced wounds in dogs [36]. The technique of freeze-drying is a commonly used method to improve the stability of tissue regeneration proteins and prolong their preservation [35]. This technique not only enhances the stability and storage potential of proteins but also retains their original biological activity. These properties make freeze-dried proteins an ideal choice for therapeutic applications. Therefore, based on these findings, the platelet dry powder we have prepared also holds potential for application in these fields, demonstrating its value in promoting wound healing and tissue regeneration. This further underscores the clinical significance and potential application scope of our research findings.
Using pig blood to mass-produce platelet dry powder can yield many benefits. In addition to a steady supply, there are also economic benefits—a byproduct of the slaughter process can be transformed into high-value biomedical products, increasing the economic value of pig blood and reducing raw material costs. Platelet dry powder can also be preserved for a long time. At normal temperatures, preparing platelet dry powder from pig blood can ensure long-term storage and immediate availability when necessary, reducing the reliance on fresh blood and enhancing the convenience of platelet dry powder for medical centers and professionals in unseen ways. Furthermore, a heightened environmental consciousness has increased the appeal of preparing platelet dry powder from pig blood. Instead of viewing pig blood as waste, this type of resource recycling contributes to environmental protection and complies with modern concepts of a green, circular economy. Last, as pig blood is rich in platelets and GFs, platelet dry powder prepared from such sources is high in quality and concentrations of both properties. Therefore, using pig blood to prepare platelet dry powder is undoubtedly a strategy with many advantages that will promote its widespread usage in biomedicine.

5. Conclusions

This study succeeded in proving that pig blood can be used in the industrial mass-production of platelet dry powder, overcoming the limitations of conventional PRP and PRF preparations that preclude them from mass-production. The results showed that the platelet dry powder prepared in this study has high concentrations of TGF-β1 and PDGF-BB that stayed within a stable range even after one year of storage. This demonstrated shelf stability may enhance the competitiveness of platelet dry powder products and their storage.

Author Contributions

Conceptualization, S.-H.L. and H.-W.F.; methodology, S.-H.L.; validation, S.-H.L.; formal analysis, S.-H.L.; investigation, S.-H.L.; data curation, S.-H.L., C.-Y.S. and H.-W.F.; writing—original draft preparation, S.-H.L.; writing—review and editing, C.-Y.S. and H.-W.F.; funding acquisition, C.-Y.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Science and Technology (MOST), Taiwan, under the grant number 111-2622-E-027-003.

Data Availability Statement

The data presented in this study are available on request from the corresponding authors. The data is not being disclosed as we are currently assessing a patent application.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Effects of calcium chloride solution concentration on platelet concentration.
Figure 1. Effects of calcium chloride solution concentration on platelet concentration.
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Figure 2. Effects of calcium chloride solution percentages on platelet concentration.
Figure 2. Effects of calcium chloride solution percentages on platelet concentration.
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Figure 3. (a,b) Effects of calcium chloride concentrations on GFs, representing the platelet dry powders prepared with different concentrations of calcium chloride, which were 0.01 M, 0.001 M, 0.0001 M, and 0.00001 M. (a) shows that the samples with 0.01 M and 0.0001 M concentrations had TGF-β1 concentrations in excess of 100,000 pg/mL, significantly higher than the other sample groups; (b) the PDGF-BB concentration was significantly higher in the sample group with the 0.001 M concentration of calcium chloride, as high as 88,000 pg/mL (averages ± SD, n = 3, * p < 0.05).
Figure 3. (a,b) Effects of calcium chloride concentrations on GFs, representing the platelet dry powders prepared with different concentrations of calcium chloride, which were 0.01 M, 0.001 M, 0.0001 M, and 0.00001 M. (a) shows that the samples with 0.01 M and 0.0001 M concentrations had TGF-β1 concentrations in excess of 100,000 pg/mL, significantly higher than the other sample groups; (b) the PDGF-BB concentration was significantly higher in the sample group with the 0.001 M concentration of calcium chloride, as high as 88,000 pg/mL (averages ± SD, n = 3, * p < 0.05).
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Figure 4. Effects of calcium chloride percentages on GFs, representing the platelet dry powders prepared with different percentages (1%, 2%, 5%, 10%, and 20%) of the calcium chloride solution. (a) shows that the addition of 20% calcium chloride solution led to TGF-β1 concentrations exceeding 100,000 pg/mL, significantly higher than the other sample groups; (b) the PDGF-BB concentration was significantly higher in the sample group with 10% calcium chloride solution, exceeding 90,000 pg/mL (averages ± SD, n = 3, * p < 0.05).
Figure 4. Effects of calcium chloride percentages on GFs, representing the platelet dry powders prepared with different percentages (1%, 2%, 5%, 10%, and 20%) of the calcium chloride solution. (a) shows that the addition of 20% calcium chloride solution led to TGF-β1 concentrations exceeding 100,000 pg/mL, significantly higher than the other sample groups; (b) the PDGF-BB concentration was significantly higher in the sample group with 10% calcium chloride solution, exceeding 90,000 pg/mL (averages ± SD, n = 3, * p < 0.05).
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Figure 5. Reproducibility across different batches, showing the GF concentrations of 6 batches of platelet dry powder prepared with the same conditions. (a) depicts a mean TGF-β1 concentration of 129,080 pg/mL; (b) shows the mean PDGF-BB concentration to be 81,173 pg/mL. Variation between batches is not substantial, demonstrating the stability of the preparation process and the repeatability of the experiment.
Figure 5. Reproducibility across different batches, showing the GF concentrations of 6 batches of platelet dry powder prepared with the same conditions. (a) depicts a mean TGF-β1 concentration of 129,080 pg/mL; (b) shows the mean PDGF-BB concentration to be 81,173 pg/mL. Variation between batches is not substantial, demonstrating the stability of the preparation process and the repeatability of the experiment.
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Figure 6. Shelf-life testing.
Figure 6. Shelf-life testing.
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Lin, S.-H.; Su, C.-Y.; Fang, H.-W. Industrial Mass Production of Platelet Dry Powder. Appl. Sci. 2023, 13, 12870. https://doi.org/10.3390/app132312870

AMA Style

Lin S-H, Su C-Y, Fang H-W. Industrial Mass Production of Platelet Dry Powder. Applied Sciences. 2023; 13(23):12870. https://doi.org/10.3390/app132312870

Chicago/Turabian Style

Lin, Shih-Hung, Chen-Ying Su, and Hsu-Wei Fang. 2023. "Industrial Mass Production of Platelet Dry Powder" Applied Sciences 13, no. 23: 12870. https://doi.org/10.3390/app132312870

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