Platelet-rich plasma-based bioactive membrane as a new advanced wound care tool.

Chronic skin ulcers, consequence of diabetes and other pathological conditions, heavily compromise the patient life quality and represent a high and constantly growing cost for National Health Services. Autologous platelet-rich plasma (PRP), has been proposed to treat these lesions. The absence of guidelines for the PRP production and the need of a fresh preparation for each treatment lead us to develop a protocol for the production of an allogenic PRP-based bioactive membrane (BAM), standardized for platelet concentration and growth factor release. This work compares BAMs obtained starting from two different platelet concentrations. There was no direct correlation between the amount of growth factors released by BAM in vitro and the initial platelet count. However, different release kinetics were noticed for different growth factors, suggesting that they were differently retained by the two BAMs. The angiogenic potential of both BAMs was determined by Luminex Angiogenesis Assay. The biological activity of the factors released by the two BAMs was confirmed by cell proliferation and migration. A diabetic mouse chronic ulcer model was used to define the best PRP therapeutic dose in vivo. Both BAMs induced wound healing by increasing the thickness of the regenerated epidermis and the vessel number. However, a too high platelet concentration resulted in a slowdown of the membrane resorption that interfered with the skin healing. Overall, the results indicate that the BAMs could represent a natural and effective wound healing tool for the treatment of skin ulcers. Copyright © 2016 John Wiley & Sons, Ltd.

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support.

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

Clinical effectiveness of platelets in additive solution treated with two commercial pathogen-reduction technologies.

BACKGROUND: Two noninferiority, randomized, controlled trials were conducted in parallel comparing the safety and efficacy of platelets treated with Intercept or Mirasol pathogen-reduction technologies versus standard platelets. STUDY DESIGN AND METHODS: The primary endpoint was the percentage of hematology patients who developed World Health Organization Grade 2 or greater bleeding. A noninferiority margin of 11% was chosen based on expected Grade 2 or greater bleeding in 20% of controls. The study was closed for financial restrictions before reaching the planned sample size of 828 patients, and an intention-to-treat analysis was conducted on 424 evaluable patients. RESULTS: In the Intercept trial (113 treated vs. 115 control patients), the absolute risk difference in Grade 2 or greater bleeding was 6.1%, with an upper one-sided 97.5% confidence limit of 19.2%. The absolute risk difference in the Mirasol trial (99 treated vs. 97 control patients) was 4.1%, and the upper one-sided 97.5% confidence limit was 18.4%. Neither absolute risk difference was statistically significant. In both trials, posttransfusion platelet count increments were significantly lower in treated versus control patients. Mean blood component use in treated patients versus controls was 54% higher (95% confidence interval, 36%-74%; Intercept) and 34% higher (95% confidence interval, 16%-54%; Mirasol) for platelets and 23% higher (95% confidence interval, 8%-39%; Intercept) and 32% higher (95% confidence interval, 10%-57%; Mirasol) for red blood cells. Unexpected reactions and adverse events were not reported. Mortality did not differ significantly between treated and control patients. CONCLUSION: Although conclusions on noninferiority could not be drawn due to low statistical power, the study provides additional information on the safety and efficacy of pathogen-reduced platelets treated with two commercial pathogen-reduction technologies.

Granulocytes and monocytes apheresis induces upregulation of TGFß1 in patients with active ulcerative colitis: A possible involvement of soluble HLA-I.

Granulocyte and monocyte apheresis has been used in different immune-mediated disorders, mainly inflammatory bowel diseases. The removal of activated leukocytes and several additional immunomodulatory mechanisms have been so far suggested to explain the anti-inflammatory effects of the treatment. Recent data indicate that, during centrifugation based apheresis, sHLA-I adsorbed to plastic circuits is able to induce TGFß1 production in activated leukocytes. On these bases, the present study was aimed at analyzing if this model could be applied to a noncentrifugation based apheresis, such as granulocyte and monocyte apheresis. Ten patients with ulcerative colitis were enrolled. Every patient received 5 weekly apheresis treatments. Cellulose acetate beads removed from the column post-GMA were stained by fluorescent anticlass I mAb and examined by fluorescent microscope. Moreover, sFasL plasma concentration, TGFß1 plasma levels, and the percentage of TGFß1 positive neutrophils were evaluated before and immediately after each single apheresis. Immunofluorescent images revealed a homogeneous layer of a sHLA-I adsorbed to the surface of the beads recovered following the procedure. sFasL plasma concentration progressively increased both following the procedures and during inter-procedure periods. Consistently, also TGFß1 plasma levels and the percentage of TGFß1 positive neutrophils increased during the procedures with a meaningful relationship with sFasL plasma levels. Taken together, these findings suggest that the immunosuppressive effects attributed to granulocyte and monocyte apheresis might depend, at least in part, on the sensitivity of activated leucocytes to the bioactivity of sHLA-I molecules. J. Clin. Apheresis 32:49-55, 2017. © 2016 Wiley Periodicals, Inc.

Combined platelet and plasma derivatives enhance proliferation of stem/progenitor cells maintaining their differentiation potential.

BACKGROUND AIMS: Platelet derivatives have been proposed as alternatives to animal sera given that for cell therapy applications, the use of fetal bovine/calf serum (FBS/FCS) is subjected to severe limitations for safety and ethical concerns. We developed a cell culture medium additive obtained by the combination of two blood-derived standardized components. METHODS: A platelet lysate (PL) and a platelet-poor plasma (PPP) were produced in a lyophilized form. Each component was characterized for its growth factor content (platelet-derived growth factor-BB/vascular endothelial growth factor). PL and PPP were used as single components or in combination in different ratio at cumulative 5% final concentration in the culture medium. RESULTS: The single components were less effective than the component combination. In primary cell cultures (bone marrow stromal cells, adipose derived adult stem cells, osteoblasts, chondrocytes, umbilical cord-derived mesenchymal stromal cells, lymphocytes), the PL/PPP supplement promoted an increased cell proliferation in respect to the standard FCS culture in a dose-dependent manner, maintaining the cell functionality, clonogenicity, phenotype and differentiative properties throughout the culture. At a different component ratio, the supplement was also used to support proliferation of a cell line (U-937). CONCLUSIONS: The PL/PPP supplement is an efficient cell culture medium additive that can replace FCS to promote cell proliferation. It can outdo FCS, especially when adopted in primary cultures from tissue biopsies. Moreover, the dual component nature of the supplement allows the researcher to determine the more appropriate ratio of the two components for the nutritional and functional requirements of the cell type of interest.

Regenerative medicine for the treatment of Teno-desmic injuries of the equine. A series of 150 horses treated with platelet-derived growth factors.

BACKGROUND/AIM: The aim of the present study was to evaluate the safety and the clinical outcome of platelet-rich plasma for the treatment of teno-desmic injures in competition horses. PATIENTS AND METHODS: From January 2009 to December 2011, 150 sport horses suffering from teno-desmic injuries were treated with no-gelled platelet-concentrate. RESULTS: No horse showed any major adverse reaction as a result of the procedure. Full healing was obtained for 81% of the horses. Twelve percent had clinical improvement and only 7% a failure. Eight percent of cases of relapse were observed. No statistically significant correlation existed between clinical outcome and the area of the lesion. A statistically significant correlation existed between the clinical outcome and the age of the horse. CONCLUSION: Treatment with platelet-derived growth factors leads to the formation of a tendon with normal morphology and functionality, which translate in the resumption of the agonistic activity for the horses we treated.

The use of platelet-rich plasma gel in patients with mixed tumour undergoing superficial parotidectomy: a randomized study.

BACKGROUND/AIM: Salivary gland tumors are mostly benign tumors. Whether a more conservative surgical approach at greater risk of recurrence, or a more radical intervention with an increased risk of facial paralysis is warranted is still under discussion. Our study addresses the opportunity for improving surgical outcome by employing platelet-rich plasma (PRP) gel at the surgical site. PATIENTS AND METHODS: Twenty consecutive patients undergoing superficial parotidectomy were randomized and assigned to two groups, one with and one without PRP gel. Many parameters were evaluated after surgery and during follow-up, such as the duration of hospitalization, facial nerve deficit, onset of Frey's syndrome, relapse, cosmetic results, presence of keloid or scar depressions, behavior of several facial muscles. RESULTS: Our explorative analysis suggests a positive effect of PRP on surgical outcome in patients undergoing parotidectomy, whereas no negative effects were detected. CONCLUSION: This work suggests that administration of PRP in patients undergoing parotidectomy is beneficial.

Biological activity of a standardized freeze-dried platelet derivative to be used as cell culture medium supplement.

Serum of animal origin and in particular fetal bovine serum is the most commonly utilized cell culture medium additive for in vitro cell growth and differentiation. However, several major concerns have been raised by the scientific community regarding the use of animal sera for human cell-based culture applications. Among the possible alternatives to the animal serum, platelet-derived compounds have been proposed since more than 10 years. Nevertheless, the high degree of variability between the different platelet preparations, and the lack of standardized manufacturing and quality control procedures, made difficult to reach a consensus on the applicability of this novel cell culture medium supplement. In this study, we describe the preparation of a standardized platelet-rich plasma (PRP) derivative obtained starting from human-certified buffy coat samples with a defined platelet concentration and following protocols including also freeze-drying, gamma irradiation and biological activity testing prior the product release as cell culture medium additive. Biological activity testing of the different preparations was done by determining the capability of the different PRP preparations to sustain human bone marrow mesenchymal stem cell (MSC) clone formation and proliferation. Taking advantage of a developed MSC in vitro clonogenicity test, we also determined biological activity and stability of the freeze-dried gamma-sterilized PRP preparations after their storage for different times and at different temperatures. The PRP effects on cell proliferation were determined both on primary cell cultures established from different tissues and on a cell line. Results were compared with those obtained in "traditional" parallel control cultures performed in the presence of bovine serum [10% fetal calf serum (FCS)]. Compared to FCS, the PRP addition to the culture medium increased the MSC colony number and average size. In primary cell cultures and in cell line cultures, the PRP promoted cell proliferation also in conditions where the FCS had not a proliferation stimulating effect due to either the nature of the cells and the tissue of origin (such as human articular chondrocytes from elderly patients) or to the critical low density cell seeding (such as for HeLa cells). In summary, the standardized PRP formulation would provide an "off-the-shelf" product to be used for the selection and expansion of several cell types also in critical cell culture conditions.

Dual effect of platelet lysate on human articular cartilage: a maintenance of chondrogenic potential and a transient proinflammatory activity followed by an inflammation resolution.

Platelet-rich plasma (PRP), a cocktail of platelet growth factors and bioactive proteins, has been proposed as a therapeutic agent to restore damaged articular cartilage. We report the biological effect of the platelet lysate (PL), a PRP derivative, on primary human articular chondrocytes cultured under both physiological and inflammatory conditions. When added to the culture medium, PL induced a strong mitogenic response in the chondrocytes. The in vitro expanded cell population maintained a chondrogenic redifferentiation potential as revealed by micromass culture in vitro and ectopic cartilage formation in vivo. Further, in chondrocytes cultured in the presence of the proinflammatory cytokine interleukin-1α (IL-1α), the PL induced a drastic enhancement of the synthesis of the cytokines IL-6 and IL-8 and of neutrophil-gelatinase associated lipocalin, a lipocalin expressed during chondrocyte differentiation and inflammation. These events were mediated by the p38 MAP kinase and NF-κB pathways. We observed that inflammatory stimuli activated phospo-MAP kinase-activated protein kinase 2, a direct target of p38. The proinflammatory effect of the PL was a transient phenomenon; after an initial upregulation, we observed significant reduction of the NF-κB activity together with the repression of the inflammatory enzyme cyclooxygenase-2. Moreover, the medium of chondrocytes cultured in the simultaneous presence of PL and IL-1α, showed a significant enhancement of the chemoattractant activity versus untreated chondrocytes. Our findings support the concept that the platelet products have a direct beneficial effect on articular chondrocytes and could drive in sequence a transient activation and the resolution of the inflammatory process, thus providing a rational for their use as therapeutic agents in cartilage inflammation and damage.

Regenerative Surgery for the Definitive Repair of a Vasculitic Nonhealing Ulcer Using Platelet-derived Growth Factors and Noncultured Autologous Cell Suspension.

SUMMARY: Vasculitic ulcers are caused by numerous disorders and may be chronic if not well treated. Various modalities of treatment, both medical and surgical, are available. We describe the case of a 63-year-old patient with a vasculitic ulcer treated with platelet-derived growth factors and noncultured autologous cell suspension collected by an innovative single-use device (ReCell).

Association of vacuum-assisted closure and platelet gel for the definitive surgical repair of an enterocutaneous fistula: a case report.

BACKGROUND: Regenerative surgery deals with damaged tissue via endogenous cell activation or through autologous cell implantation. Several clinical applications employing cell infusions, platelet gel (PG), or both, are currently applied in cases in which no other therapy is application. The vacuum-assisted closure (VAC) system is a non invasive device used in the management of complicated wounds, which creates sub-atmospheric pressure promoting the wound healing process. PATIENTS AND METHODS: We describe the case of a 75-year-old woman who underwent several surgical interventions and presented a non-healing ileo-cutaneous fistula. All standard procedures in order to treat the trauma failed, so a treatment associating VAC and PG was performed. DISCUSSION AND CONCLUSION: VAC and PG represent promising opportunities for the treatment of difficult wounds. In this case, the association of regenerative medicine using homologous PG to the VAC therapy was employed in order to enhance the effect of both techniques on tissue repair.

Tension-free primary closure with autologous platelet gel versus Vivostat- for the definitive treatment of chronic sacrococcygeal pilonidal disease.

OBJECTIVE: A randomized clinical trial was performed in patients with chronic or recurrent pilonidal sinus (PS) comparing primary closure coupled with random application of in house autologous platelet gel or produced by means of Vivostat- in order to assess whether a standardized product had an impact on the wound healing process. PATIENTS AND METHODS: Between June 2006 and June 2009, 100 patients (82 males, 18 females: median age 30 years; range, 16-51 years) underwent wide excision of the pilonidal area with midline tension-free closure and were randomly given either the in house autologous platelet gel (Group 1) or the Vivostat- gel (Group 2). RESULTS: Group 2 patients had shorter wound healing time (8 vs. 10 days; p<0.0001), time to return to full activity (11 vs. 16 days: p<0.0001), less uncomplicated fluid collections (120 vs. 190 ml: p<0.0001), and fewer postoperative wound complications (1/50=2% vs. 5/50=10%, p<0.001). After a median follow-up of 21 months (range: 4-40 months), two recurrences were detected in Group 1. CONCLUSION: The standardized production of platelet gel by means of the Vivostat- system guarantees the reproducibility of the procedure and its use was correlated with an improved outcome, with a high degree of patient satisfaction and better cosmetic results.

Regenerative medicine for the definitive surgical repair of pilonidal sinus. A new method of wound reconstruction.

OBJECTIVE: A retrospective analysis of patients undergoing surgery for complex (> or =3 tracks) or recurrent pilonidal sinus (PS) was performed; the results of this clinical experience were compared with an original method of primary wound closure, coupling a "tension-free" technique of wound reconstruction with autologous cryoplatelet gel application, in order to improve the wound healing process and reduce the postoperative disability period. PATIENTS AND METHODS: The retrospective analysis included 30 patients undergoing surgery for PS between January 2003 and May 2005: in the first group of 15 consecutive patients, the wound was left open to close secondarily while in the remaining 15 patients, primary closure by means of a "tension-free" technique of wound reconstruction was attempted. Between June 2005 and May 2006, another subset of 15 patients was prospectively recruited, coupling the "tension-free" technique of wound reconstruction with autologous cryoplatelet gel application. RESULTS: In the first group of patients, median postoperative disability accounted for 65 days with one recurrence. In the second group, primary healing was achieved in 11 patients, with a median postoperative disability of 28 days; two recurrences did occur. In the third group of patients, primary healing was achieved in all patients within 14 to 29 days, and no recurrence has yet been detected. CONCLUSION: Short follow-up notwithstanding, the simplicity of the operation, the use of autologous products and the minimal postoperative disability with complete wound healing suggest that this new approach may represent a useful alternative to current surgical techniques for PS excision.

Clinical applications of autologous cryoplatelet gel for the reconstruction of the maxillary sinus. A new approach for the treatment of chronic oro-sinusal fistula.

The authors report their clinical experience regarding an original method of surgical repair of oro-sinusal communications. From September 1999 to December 2003, 13 patients (7 male and 6 female patients; mean age: 52 years, range: 24-68 years) underwent surgical repair of an oro-antral fistula by means of cryoplatelet gel: in three patients, it was mixed with bioglass granules; in two, it was mixed with Bioss; in three, it was mixed with particulate bone extracted by means of a bone grafter from the oral cavity close to the operative site, with addition of demineralised bovine bone; in three, it was used together with porose hydroxyapatite, and in two patients the cryoplatelet gel was used only. No postoperative complication was reported; primary wound healing was achieved within seven to nine days. A bony orthopantoscintigraphy was performed a few months following the operative procedure, showing an active osteogenic process. In eight patients, a CT was performed after 8 to 12 months from the operation, showing a normal pneumatization with reconstruction of the floor of the maxillary sinus. Although preliminary, these findings seem to suggest that the use of bioengineered materials coupled with growth factors and osteoprogenitor cells may represent a valuable alternative to autologous bone transplantation for the reconstruction of the maxillary sinus.

Guidelines for the clinical use of albumin: comparison of use in two Italian hospitals and a third hospital without guidelines.

BACKGROUND: In the absence of clinical practice guidelines prior to 1999, the consumption of human albumin in the Liguria region of Italy was very high, despite possible adverse effects, limited supply, and significant cost. OBJECTIVE: The purpose of this study was to assess the impact of comprehensive guidelines on the amount of albumin used in 2 general hospitals and to compare it with that of a third general hospital that did not adopt the guidelines. METHODS: We analyzed the influence of the guidelines on albumin use in 2 general hospitals (hospitals 1 and 2) in the Liguria region by comparing albumin consumption during the year before the distribution of the guidelines (1999) with consumption in the 2 years after their distribution (2000 and 2001). We compared these data with those of a third general hospital that did not adopt the guidelines (hospital 3). The parameters considered were total consumption of albumin, consumption per bed, consumption per hospital stay, mean time to discharge, expenditure per bed, and mortality rate. RESULTS: In the years 2000 and 2001, the adoption of guidelines reduced albumin consumption in hospitals 1 and 2. In hospital 1, where the release of albumin was carefully controlled by the transfusion service, albumin use per hospital stay decreased 8.7% in 2000 and 7.6% in 2001 from 1999; in hospital 2, use decreased 73.8% and 77.4%, respectively, from 1999. In hospital 3, rejection of the guidelines was coupled with an increase of 2.9% and 8.4%, respectively, in the amount of albumin used per hospital stay. In the years 2000 and 2001, the savings in the expenditure for albumin was ∼17,000 euro in hospital 1 and ∼200,000 euro in hospital 2. CONCLUSION: This study confirms that the adoption of guidelines may substantially reduce the inappropriate use of albumin and relative costs.