Tomasz Bielecki

Antibacterial effect of autologous platelet gel enriched with growth factors and other active substances AN IN VITRO STUDY

Platelet-rich plasma is a new inductive therapy which is being increasingly used for the treatment of the complications of bone healing, such as infection and nonunion. The activator for platelet-rich plasma is a mixture of thrombin and calcium chloride which produces a platelet-rich gel. We analysed the antibacterial effect of platelet-rich gel in vitro by using the platelet-rich plasma samples of 20 volunteers. In vitro laboratory susceptibility to platelet-rich gel was determined by the Kirby-Bauer disc-diffusion method. Baseline antimicrobial activity was assessed by measuring the zones of inhibition on agar plates coated with selected bacterial strains. Zones of inhibition produced by platelet-rich gel ranged between 6 mm and 24 mm (mean 9.83 mm) in diameter. Platelet-rich gel inhibited the growth of Staphylococcus aureus and was also active against Escherichia coli. There was no activity against Klebsiella pneumoniae, Enterococcus faecalis, and Pseudomonas aeruginosa. Moreover, platelet-rich gel seemed to induce the in vitro growth of Ps. aeruginosa, suggesting that it may cause an exacerbation of infections with this organism. We believe that a combination of the inductive and antimicrobial properties of platelet-rich gel can improve the treatment of infected delayed healing and nonunion.

Benefit of Percutaneous Injection of Autologous Platelet-Leukocyte-Rich Gel in Patients with Delayed Union and Nonunion

This article reports the efficacy of percutaneous autologous platelet-leukocyte-rich gel (PLRG) injection as a minimally invasive method alternative to open grafting techniques. Each of 32 participants was followed on a regular basis with clinical examinations, roentgenograms, dual-energy X-ray absorptiometry examinations. In the delayed union group, the average time to union was 9.3 weeks after PLRG injection and the union was achieved in all cases. In the nonunion group, the union was observed in 13 of 20 cases and the average time to union was 10.3 weeks after PLRG injection. Interestingly, in patients in whom union was not achieved, the average time from the fracture and/or from the last operation was >11 months. This is our initial experience with the use of PLRG as biologic treatment for delayed union or nonunion. Our investigation showed that percutaneous PLRG injection in delayed union is a sufficient method to obtain union, which is less invasive procedure than bone marrow injection. Percutaneous PLRG grafting can be also an effective method for the treatment of selected cases of nonunion. The essential factor is the average time from the initial surgery to PLRG injection for nonunion; <11 months seems to be critical for good outcomes.

Shedding light in the controversial terminology for platelet-rich products: Platelet-rich plasma (PRP), platelet-rich fibrin (PRF), platelet-leukocyte gel (PLG), preparation rich in growth factors (PRGF), classification and commercialism

A recent series of letters were published in JBMR-A about platelet concentrates for surgical use, where both terminology and content of these materials were hotly debated. The definition and classification of the platelet concentrate products are indeed very important issues, as many misunderstandings are widely spread in the large literature on this topic. These techniques were initially gathered under the name ‘‘platelet-rich plasma (PRP),’’ in reference to the generic term used in transfusion hematology, but this name is too general for the qualification of the many products developed now. In the first letter, Everts et al. insisted on the presence of leukocytes in most platelet preparations for surgical use. These authors explained a very important truth that many PRPs were in fact leukocyteand platelet-rich plasmas (LPRPs), and that the presence of leukocytes in these surgical adjuvants may be highly beneficial. They thus, introduced the term of ‘‘platelet-leukocyte–rich plasma (P-LRP).’’ Moreover, they pointed out that the two activation forms of the product (liquid platelet suspension or gelified fibrin-platelet clot) have different characteristics, and that the concentrates activated with a fibrinogen-cleaving agent (thrombin, batroxobin) should be named in fact as ‘‘platelet-leukocyte gels (PLG).’’ In this letter, these authors resumed the clarification process of the platelet concentrate definitions started in 2006. However, their proposals for terminology were not complete and have been improved and systematized in the recent publication of a wide classification system for these products. The first concern is that all PRPs do not contain leukocytes. Many PRPs obtained from cell separator units or from the Anitua’s preparation rich in growth factors (PRGF) subfamilies do not contain leukocytes and were classified as pure PRP (P-PRP). On the contrary, PRPs containing leukocytes were classified as L-PRP: this acronym seems obviously more logical and reader-friendly than P-LRP, but we agree that a consensus should be found to solve this issue once for good. The second issue is related to the gel form terminology. ‘‘Platelet gel’’ and ‘‘PLG’’ are too general terms. Indeed, products with a high-density fibrin network also exist and were classified as ‘‘platelet-rich fibrin (PRF),’’ some with leukocytes [leukocyteand platelet-rich fibrin (L-PRF)] and some without leukocytes [pure platelet-rich fibrin (P-PRF)]. All these PRFs are only available in the form of a very dense fibrin gel, while PRP gels are never so strong and dense. We thus believe that the activated form of P-PRP or L-PRP should simply be named ‘‘P-PRP gel’’ and ‘‘L-PRP gel’’ to differentiate them from the products of the PRF families. In the second letter, Anitua et al. agreed that the recent development of many different techniques with various platelet and leukocyte contents led to a confusing jungle of terms and products. This notion of ‘‘jungle of platelet concentrates’’ was already pointed out some years ago, when the main confusion between PRPs and the first PRF appeared. Anitua et al. were right in their call for the definition of a relevant terminology but their approach was unfortunately partisan. First, Anitua et al. claimed that leukocytes should be avoided in platelet concentrates for surgical use, to avoid the proinflammatory effects of the proteases and acid hydrolases contained in white blood cells, particularly when injected in tendons. However, these authors did not justify their statement with scientific evidence; to sustain their claim, Anitua et al. cited Ref. 9 describing very positive anabolic effects on tendon cells obtained with a PRP, . . . but the PRP described in this study was in fact a leukocyte-rich PRP. This question of the leukocyte content within platelet concentrates for surgical use is in fact an old debate. There is however actually no proof that the leukocytes within these surgical preparations might have undesirable side effects. On the contrary, several studies showed that L-PRPs have antimicrobial effects, but no undesirable inflammatory reactions have been observed with L-PRPs

Autologous platelets and leukocytes can improve healing of infected high-energy soft tissue injury

Despite advances in surgical techniques and pharmacology, healing of injury-associated soft tissue defects is frequently complicated by infections, which often requires a secondary intervention. Cytokines are important mediators of healing. Application of autologous platelet-rich plasma enriched in growth factors and antimicrobial proteins, known also as platelet-leukocyte rich plasma (PLRP), represents a novel approach to the treatment of soft tissue and bone healing disturbances. This case is the first report on the application of PLRP in an infected high-energy soft tissue injury and shows that the volume and concentration of platelets and leukocytes is adequate to induce healing processes despite concurrent infection.

Improved treatment of mandibular odontogenic cysts with platelet-rich gel

BACKGROUND Platelets play a central role in hemostasis and healing processes. By concentrating platelets, platelet-rich plasma (PRP) with higher levels of growth factors can be obtained, which might stimulate healing processes. After platelet degranulation, massive release of growth factors and active substances occurs and gelatinous matrix-platelet-rich gel (PRG) is formed. OBJECTIVE In this double-blind study, we report the influence of PRG on healing of mandibular odontogenic cysts. STUDY DESIGN We examined 23 cases divided into control (no PRG treatment) and experimental (PRG-treated) groups. Each participant was followed on a regular basis with clinical examinations, roentgenograms, and dual-energy x-ray absorptiometry (DEXA) examinations. RESULTS Clinical observations showed that oral mucosa healed faster in patients treated with PRG compared with cases where gel was not added. Roentgenograms and DEXA examinations showed considerable enhancement of bone regeneration beginning from the 5th week and continuing during subsequent periods after implantation of PRG in the experimental group compared with the control group. CONCLUSIONS In our opinion, PRG possesses inductive properties that could stimulate the healing processes. The use of autologous growth factors from platelet gel seems to be one of the most promising methods of treatment of bone, cartilage, and soft tissue defects.

A side-effect induced by the combination of a demineralized freeze-dried bone allograft and leucocyte and platelet-rich plasma during treatment for large bone cysts: a 4-year follow-up clinical study.

During platelet activation over 30 growth factors are released from their alpha granules. By concentrating platelets, higher levels of growth factors are obtained and can be used to stimulate the healing processes. Moreover, during blood centrifugation, huge numbers of leukocytes and thrombocytes can be obtained to form leukocyte and platelet-rich plasma (L-PRP). After platelet degranulation the active gelatinous matrix called leukocyte and platelet-rich gel (L-PRG) is formed. In the present clinical study, we report the influence of PLRG and freeze-dried allografts on healing of large femoral bone cysts. Each of 6 participants was followed on a regular basis with clinical examinations, roentgenograms and dual-energy X-ray absorptiometry (DXA) examinations. Our four-year follow up clinical study using allografts and PLRG in the treatment of large cystic lesions of the proximal femur showed that mixing these substances is not efficient and it might induce unknown local reactions between them causing complete bone graft destruction. Further studies are necessary to fully understand the mechanisms of the local activity of L-PRP gel.

A review of decellurization methods caused by an urgent need for quality control of cell-free extracellular matrix' scaffolds and their role in regenerative medicine: REVIEW OF DECELLURIZATION METHODS

The natural extracellular matrix (ECM),thanks to its specific properties (e.g., collagenous lattice, a reservoir of growth factors, ECM-cell anchoring areas, an optimal pH and CO2 ),ensures an optimal microenvironment for homeostatic and regenerative cell development. In the context of regenerative medicine, ECM is a lair for residual and infiltrative cells. The aim of the clinical usage of cell-free ECM scaffolds is the enhancement of tissue regeneration with possible minimization of an adverse host reaction on allogeneic or xenogeneic biomaterial. Thus, the objective of decellularization is to obtain acellular grafts characterized by optimal biological properties, such as a lack of remaining cellular elements (e.g., cell membrane phospholipids and proteins, nucleic acids, mitochondria), lack of immunogenicity, lack of calcification promotion and lack of cytotoxicity (e.g., in unrinsed detergents). Furthermore, cell-free ECM scaffolds should present the optimal mechanical and structural properties that may ensure the biocompatibility of the graft. The maintenance of the ultrastructure composition of the ECM is one of the most important goals of decellularization. All physical, chemical, and biological methods proposed (used separately or in combination to extract cells from tissues/organs) are not 100% effective in cell removal and always cause a disruption of the ECM texture, as well as a probable loss of important structure components. Although cell-free ECM scaffolds are generally classified as medical devices, there are no widely accepted or legally defined criteria for quality control/evaluation methods of obtained matrices. Such criteria must be provided. Some of them have been proposed in this manuscript.

Benefit of Leukocyte- and Platelet-Rich Plasma in Operative Wound Closure in Oral and Maxillofacial Surgery

This article reports the influence of an autologous leukocyte- and platelet-rich plasma (L-PRP) injection as a minimally invasive method on supporting wound healing processes after a mandibular odontogenic cystectomy and double mandibular fracture fixation. 113 patients were enrolled into a control group (received no L-PRP injection) and 102 patients were enrolled into an L-PRP group with an oral mucosa incision. 18 patients after a double mandibular fracture were operated on using 2 external submandibular approaches receiving no fluids in the right site (a control group) and an L-PRP injection in the left incision (L-PRP group). Clinical observations showed that the oral mucosa healed faster in patients treated with L-PRP, in comparison to cases where inductive biomaterial was not added. Pain at the L-PRP injection site was relieved within few hours after an operation in patients with double mandibular fractures. However, there were no differences observed in the progression of the healing process. L-PRP possesses inductive properties that could stimulate healing processes and it seems to be one of the most promising methods in the future for the treatment of soft tissue defects.

A new aspect of in vitro antimicrobial leukocyte- and platelet-rich plasma activity based on flow cytometry assessment

Abstract The current literature suggests that the antibacterial effect of leukocyte- and platelet-rich plasma (L-PRP) is directly related to platelet and leukocyte concentrations. The aim of this study was twofold: first, to evaluate the antimicrobial effect of L-PRP against selected bacterial strains in vitro, and second, to correlate this effect with leukocyte and platelet content in the final concentration. Blood was collected from 20 healthy males, and L-PRP, acellular plasma (AP), and autologous thrombin were consecutively prepared. Flow cytometry analysis of the blood, L-PRP, and AP was performed. The L-PRP gel, liquid L-PRP, and thrombin samples were tested in vitro for their antibacterial properties against seven selected bacterial strains using the Kirby–Bauer disk-diffusion method. There was notable antimicrobial activity against selected bacterial strains. No statistically significant correlations between antimicrobial activities and the platelet concentration in L-PRP were observed. Statistically significant positive correlations between selected leukocyte subtypes and antimicrobial activity were noted. A negative correlation was found between elevated monocyte count and antimicrobial activity of L-PRP against one bacterial strain studied. L-PRP possesses antimicrobial activity and can be potentially useful in the fight against certain postoperative infections. The bactericidal effect of L-PRP is caused by leukocytes, and there exists a relationship among selected leukocyte subtypes and L-PRP antimicrobial activity.

Abduction dislocation of the knee joint--a case study.

The paper presents a case of untypical, not included in existing classifications, knee joint dislocation in a young man. An MRI scan confirmed a rupture of both cruciate ligaments and damage to the ligamento-capsular complex on the medial side of the knee joint. Two weeks after injury, an arthroscopy was performed with joint lavage followed by repair of the damaged ligamento-capsular complex. A very good functional result was obtained three years after the injury, in spite of the patient not having consented to an elective cruciate ligament reconstruction.