Evaluation of the influence of platelet-rich plasma (PRP), platelet lysate (PL) and mechanical loading on chondrogenesis in vitro

The aim of this work is to investigate the capability of PRP as an adjuvant therapy to autologous chondrocyte implantation (ACI) in combination with multi-axial load with respect to cartilage regeneration. Articular cartilage shows poor repair capacity and therapies for cartilage defects are still lacking. Well-established operative treatments include ACI, and growing evidence shows the beneficial effects of PRP. Platelets contain numerous growth factors, among them transforming growth factor beta (TGF-β). Dynamic mechanical loading is known to be essential for tissue formation, improving extracellular matrix (ECM) production. For our ACI model monolayer expanded human chondrocytes were seeded into polyurethane scaffolds and embedded in fibrin (hChondro), in PRP-Gel (PRP), or in fibrin with platelet lysate (PL), which was added to the media once a week with a concentration of 50 vol%. The groups were either exposed to static conditions or multi-axial forces in a ball-joint bioreactor for 1 h per day over 2 weeks, mimicking ACI under physiological load. The culture medium was collected and analyzed for glycosaminoglycan (GAG), nitrite and transforming growth factor beta 1 (TGF-β1) content. The cell-scaffold constructs were collected for DNA and GAG quantification; the expression of chondrogenic genes, TGF-β and related receptors, as well as inflammatory genes, were analyzed using qPCR. Loading conditions showed superior chondrogenic differentiation (upregulation of COL2A1, ACAN, COMP and PRG4 expression) than static conditions. PRP and PL groups combined with mechanical loading showed upregulation of COL2A1, ACAN and COMP. The highest amount of total TGF-β1 was quantified in the PL group. Latent TGF-β1 was activated in all loaded groups, while the highest amount was found in the PL group. Load increased TGFBR1/TGFBR2 mRNA ratio, with further increases in response to supplements. In general, loading increased nitrite release into the media. However, over time, the media nitrite content was lower in the PL group compared to the control group. Based on these experiments, we conclude that chondrogenic differentiation is strongest when simulated ACI is performed in combination with dynamic mechanical loading and PRP-gel or PL supplementation. An inflammatory reaction was reduced by PRP and PL, which could be one of the major therapeutic effects. Loading presumably can enhance the action of TGF-β1, which was predominantly activated in loaded PL groups. The combination of load and PRP represents an effective and promising synergy concerning chondrocyte-based cartilage repair.

Role of Platelet Rich Plasma in Accelerating the Healing Rate of Diabetic Foot Ulcer

Background Diabetes is a major health problem that is currently showing an alarming rise in its prevalence, this has recently been estimated at 7.8% in the United States, presenting a > 50% increase over the past 15 years while there exists a large population group in whom diabetes is undiagnosed. Objective To evaluate the effectiveness and rate of healing of autologous PRP gel in treatment of diabetic foot ulcers. Compare the effectiveness of PRP gel with standard treatment (normal saline dressings). Methods 30 patients with non-ischemic DFU were classified into two groups: Group A: Patients with non-ischemic DFU and were treated with a novel modality i.e.: PRP injection in the healing edge and the floor of the targeted ulcer. GroupGroup B B:: Patients with non-ischemic DFU who had usual standard care i.e.: moist dressing with or without collagenase ointment. All cases had a minimal debridement prior to treatment, here in our study the demographic data i.e. Age, sex, medical history were homogenously distributed among both groups. All cases in both groups were non ischemic after successful revascularization either by OR or ER. Results The rate of complete healing for ulcers in group A was achieved in six patients (40%) at the fifth week, while five patients (33.33%) were healed completely by the sixth week and only one patient (6.67%) healed in the ninth week. 26.67% (n = 4 cases) in group B showed complete healing rate by eighth week and 40% (n = 6 cases) were healed by ninth week while 33.33% (n = 5 cases) were healed in the tenth week. P value was statistically significant <0.001 between the groups. Conclusion Activated platelet rich plasma is a novel modality in treatment of diabetic foot ulcers which is feasible, safe and effective with high rate of limb salvage rate and clinical improvement.

The effectiveness of platelet-rich plasma gel on full-thickness cartilage defect repair in a rabbit model

Aims The present study investigates the effectiveness of platelet-rich plasma (PRP) gel without adjunct to induce cartilage regeneration in large osteochondral defects in a rabbit model. Methods A bilateral osteochondral defect was created in the femoral trochlear groove of 14 New Zealand white rabbits. The right knees were filled with PRP gel and the contralateral knees remained untreated and served as control sides. Some animals were killed at week 3 and others at week 12 postoperatively. The joints were harvested and assessed by Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) MRI scoring system, and examined using the International Cartilage Repair Society (ICRS) macroscopic and ICRS histological scoring systems. Additionally, the collagen type II content was evaluated by the immunohistochemical staining. Results After 12 weeks post-surgery, the defects of the PRP group were repaired by hyaline cartilage-like tissue. However, incomplete cartilage regeneration was observed in the PRP group for three weeks. The control groups showed fibrocartilaginous or fibrous tissue, respectively, at each timepoint. Conclusion Our study proved that the use of PRP gel without any adjuncts could successfully produce a good healing response and resurface the osteochondral defect with a better quality of cartilage in a rabbit model. Cite this article: Bone Joint Res 2021;10(3):192–202.

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use

ABSTRACT: Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-β, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF-β (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.

Spinal Reflex Recovery after Dorsal Rhizotomy and Repair with Platelet-Rich Plasma (PRP) Gel Combined with Bioengineered Human Embryonic Stem Cells (hESCs)

Dorsal root rhizotomy (DRZ) is currently considered an untreatable injury, resulting in the loss of sensitive function and usually leading to neuropathic pain. In this context, we recently proposed a new surgical approach to treat DRZ that uses platelet-rich plasma (PRP) gel to restore the spinal reflex. Success was correlated with the reentry of primary afferents into the spinal cord. Here, aiming to enhance previous results, cell therapy with bioengineered human embryonic stem cells (hESCs) to overexpress fibroblast growth factor 2 (FGF2) was combined with PRP. For these experiments, adult female rats were submitted to a unilateral rhizotomy of the lumbar spinal dorsal roots, which was followed by root repair with PRP gel with or without bioengineered hESCs. One week after DRZ, the spinal cords were processed to evaluate changes in the glial response (GFAP and Iba-1) and excitatory synaptic circuits (VGLUT1) by immunofluorescence. Eight weeks postsurgery, the lumbar intumescences were processed for analysis of the repaired microenvironment by transmission electron microscopy. Spinal reflex recovery was evaluated by the electronic Von Frey method for eight weeks. The transcript levels for human FGF2 were over 37-fold higher in the induced hESCs than in the noninduced and the wildtype counterparts. Altogether, the results indicate that the combination of hESCs with PRP gel promoted substantial and prominent axonal regeneration processes after DRZ. Thus, the repair of dorsal roots, if done appropriately, may be considered an approach to regain sensory-motor function after dorsal root axotomy.

Evaluation of platelet-rich plasma gel as an angiogenesis-inducing agent in canineadvancement skin flaps

ABSTRACT: This work aimed to evaluate the effect of platelet-rich plasma (PRP) on advancement skin flaps in dogs regarding improvement of vascularization, with focus on increasing its viable area, since there are reports that it is a potential angiogenesis stimulator. The experimental group was composed of eight adult bitches, in which two advancement skin flaps were made in the ventral abdominal region. No product was applied in the control flap (CF), while PRP was used in the contralateral flap, called treated flap (TF). The areas were clinically evaluated every two days until the 7th postoperative day regarding skin color and presence of necrosis. At 10 days, both flaps were removed and submitted to histological examination and blood vessel morphometry. The vessels counted in each group were statistically analyzed by the F-test at 1% probability. Results showed no significant difference in macroscopic changes in the wound, or CF and TF vascularization, thus suggesting that PRP gel did not improve advancement skin flap angiogenesis in bitches under the experimental conditions in which this research was developed.

A possible Injectable Tissue Engineered Nucleus Pulposus constructed with platelet-rich plasma and ADSCs in Vitro

Background: Injectable tissue engineered nucleus pulposus is a new idea for minimally invasive repair for degenerative intervertebral disc. The platelet-rich plasma (PRP) and adipose derived stromal cells(ADSCs) could be harvested from autologous tissue easily. PRP has mixed autologous growth factors and fibrous reticulate structure which may have the potential to make ADSCs differentiated into nucleus pulposus-like cells. The goal of this study was to explore the feasibility of constructing a possible injectable tissue engineered nucleus pulposus with PRP gel scaffold and ADSCs. Methods: After the rabbit ADSCs were identified with flow cytometry, the ADSCs were seeded into PRP gel and cultured in vitro. At the 2nd, 4th and 8th week, the PRP gel/ADSCs complex was observed by macroscopy, histological staining, BrdU immunofluorescence, and scanning electron microscopy. The glycosaminoglycans (GAG) in the PRP gel/ADSCs complex were measured by safranin O staining with spectrophotometry. PRP gel/ADSCs complex genes expression of HIF-1α, Aggrecan, Type Ⅱ collagen were detected by RT-PCR. The injectability of this complex was tested. Results: Macroscopically, the complex was solidified into gel with smooth surface and good elasticity. The safranin O staining confirmed almost no positive staining at 2nd week, however, the positive staining of extracellular matrix was enhanced obviously at 4th, 8th week. The HE staining and SEM demonstrated that the cells were well-distributed in the reticulate scaffold. BrdU immunofluorescence showed that ADSCs can survive and proliferate in PRP gel at each time points. The level of GAG at 4th week was higher than those at 2nd week (P<0.05), and significant difference was also noted between 4th and 8th week (P<0.05). HIF-1α, Aggrecan, Type Ⅱ collagen genes expression at 4th week were much more than those at 2nd week (P<0.05), and significant differences were also noted between 4th and 8th week (P<0.05). The flow rate of complex was 0.287 mL/min when passed through the 19-gauge needle with the 100mmHg pressure. Conclusions: The PRP gel made it possible for rabbit ADSCs differentiated into nucleus pulposus-like cells after mixed culture in vitro. Maybe, it is a better feasible method for construction of autologous injectable tissue engineered nucleus pulposus.

Differences in results of infrabony pocket treatment with addition of platelet rich fibrin and platelet rich plasma gel in DFDBA bone graft

Treatment of infrabony pocket makes use of bone graft material demineralized freeze dried bone allograft (DFDBA) from different individuals which has undergone demineralization process and it is osteoinductive. Enhancement of growth factor was done by adding platelet rich fibrin (PRF) and platelet rich plasma (PRP). PRP is activated with an addition of calcium chloride CaCl2) to form gel. The method used to apply the bone grafting material is open flap debridement OFD). This research aimed to reveal the differences in the results of infrabony pocket treatment using PRF and PRP gel with an addition of DFDBA. The sample was taken from 20 infrabony pocket points divided into 2 groups, 10 infrabony pocket were treated with OFD+DFDBA+PRF and the other were treated with OFD+DFDBA+PRP gel. Pocket depth (PD) was measured on the baseline and the first and third month after treatment. Alveolar bone height was measured using cone beam computed tomography (CBCT) radiograph on the baseline to the third month after treatment. The results of this research showed that there was difference in the results of infrabony pocket treatment using PRF and PRP gel with an addition of DFDBA which could be observed from a reduction in PD from the baseline, month 1 and month 3 as well as reduction in alveolar bone height from the baseline to month 3. This research concluded that infrabony pocket treatment PRF application yields better results than PRP gel application in terms of PD and alveolar bone height reduction.