George Agrogiannis

The Influence of Platelet-Rich Plasma on Angiogenesis During the Early Phase of Tendon Healing

Background: The poor vascularity of tendons is a major factor in their limited healing capacity. The aim of this study was to assess the effect of Platelet Rich Plasma (PRP) on angiogenesis during tendon healing. Materials and Methods: Forty-eight skeletally mature New Zealand White rabbits were used. The Achilles tendon was transected transversely and 0.5 ml of PRP was injected into the tendon mass on each side of the incision on both limbs. The injection in the control group consisted of saline. Six animals from each group (12 tendons each) were sacrificed after 1, 2, 3, and 4 weeks following treatment. Three sections from each Achilles were stained with hematoxylinosin for microscopic examination. Further three sections were immunostained with a monoclonal antibody against CD31 (Daco Co), followed by image analysis to count new vessel numbers and statistical analysis was performed. Results: There was significantly more angiogenesis in the PRP group compared to the control group during the first two weeks of the healing process, i.e., inflammatory and proliferative phase (p < 0.0001). The orientation of collagen fibers in the PRP group was better organized. The number of the newly formed vessels in the PRP group were significantly reduced at 4 weeks compared to the controls (p < 0.0001) suggesting the healing process was shortened. Conclusion: PRP seems to enhance neovascularization which may accelerate the healing process and promote scar tissue of better histological quality. Clinical Relevance: Although these results need replication and further biomechanical research, PRP may promote tendon healing acceleration.

Caspase-3 immunohistochemical expression is a marker of apoptosis, increased grade and early recurrence in intracranial meningiomas

Caspase-3 is the ultimate executioner caspase that is essential for the nuclear changes associated with apoptosis. We investigated caspase-3 immunohistochemical expression in 58 primary intracranial meningiomas, using one monoclonal antibody detecting both precursor and cleaved caspase-3 (CPP32) and a second recognizing only the cleaved activated form (ASP175). Caspase-3 expression was analyzed in relation to baseline apoptosis—as illustrated by the expression of anti-single stranded DNA (ss-DNA), the antiapoptotic protein bcl-2, proliferation indices (Ki-67, PCNA, topoisomerase IIa, mitosin C), hormonal status (estrogen, progesterone, androgen receptors), standard clinicopathological parameters and patients’ disease-free survival. Caspase-3 immunostaining was observed in 62% of cases for CPP32 and in 24% for ASP175. In both instances, the labeling index (LI) was significantly correlated with ss-DNA LI (p=0.038 and p=0.018). CPP32 but not ASP175 LI positively correlated with the mitotic index (p=0.001) and PCNA LI (p=0.004). Both CPP32 and ASP175 LIs were increased in nonbenign meningiomas (p<0.0001 and p=0.0035 respectively). In univariate and multivariate survival analyses, caspase-3 predicted meningioma recurrence, independently affecting disease-free survival (p=0.011 and p=0.047 respectively for CPP32; p<0.0001 and p=0.012 respectively for ASP175). Caspase-3 may prove to be a useful predictor of early recurrence in a group of neoplasms characterized by the frequent discordance between histology and clinical behavior.

Experimental study of tendon healing early phase: Is IGF-1 expression influenced by platelet rich plasma gel?

BACKGROUND It is well established that growth factors play a critical role in the healing process of connective tissues. To our knowledge, there are no studies in literature concerning the influence of PRP on growth factors expression. HYPOTHESIS The aim of this study was to assess the effect of a single application of platelet rich plasma (PRP) gel in a patellar tendon defect on the spatial and temporal expression of Insulin-like Growth Factor 1 (IGF-1) during tendon healing. MATERIALS AND METHODS Twenty-four animals were randomized to receive PRP (PRPFast, Bioteck) in a gel form (PRP group) and 24 to serve as untreated controls (Control group). A defect of 3 mm x 10 mm was surgically created on the tendon under general anaesthetic and in the PRP group, PRP gel was applied to fill the tendon defect whereas no treatment was applied in the control group. Six animals (12 limbs) from each treatment-group were sacrificed after one, two, three and four weeks following treatment. Histological and immunohistochemical staining were performed. RESULTS Histology revealed a faster healing process in the tendons of PRP group in comparison with the controls. In the first 2 weeks of healing, IGF-1 was found intracellularly in various type cells, whereas in the last 2 weeks of healing, IGF-1 was detected mainly in tenocytes. Both cytoplasmic and nuclear expressions were present, whereas the larger amounts of immunoexpression were localized in both epitenon and endotenon. A superior expression of IGF-1 was seen in PRP group compared with controls (p<0.0001) in both the epitenon and endotenon at each time point except at 4th week of healing where a superior expression of IGF-1 was shown in the endotenon of control group, compared to the PRP group (p<0.0001). CONCLUSION PRP may improve tendon defect healing by overexpression of IGF-1. LEVEL OF EVIDENCE Laboratory control animal study.

Expression of tissue inhibitor of matrix metalloproteinases (TIMP)-3 protein in invasive breast carcinoma: Relation to tumor phenotype and clinical outcome

IntroductionOur aim was to study the expression pattern of tissue inhibitor of metalloproteinases (TIMP)-3 protein in invasive breast carcinoma, and its clinicopathological and prognostic value as well as its relation to markers indicative of the tumor phenotype.MethodsImmunohistochemistry was performed on paraffin-embedded tissue specimens from 173 invasive breast carcinomas to detect the proteins TIMP-3, estrogen receptor (ER), progesterone receptor, p53, c-erbB-2, topoisomerase IIα and Bcl-2.ResultsTIMP-3 protein was immunodetected in the cytoplasm of the malignant cells and the peritumoral stroma, as well as in in situ carcinoma and normal epithelium. Reduced expression of TIMP-3 protein within cancer cells was correlated with carcinomas of high nuclear and histological grade (p = 0.032 and p = 0.015, respectively), and low ER expression (p = 0.053). Moreover, TIMP-3 immunopositivity was inversely correlated with the expression of p53 and topoIIα proteins (p = 0.002 and p = 0.008, respectively), whereas it was positively associated with Bcl-2 expression (p = 0.020). Reduced expression of TIMP-3 protein within cancer cells was found to have an unfavorable impact on disease-free survival (p = 0.052) in the entirety of the patient population, as well as in both subgroups of lymph-node-positive and mutant-p53-negative patients (p = 0.007 and p = 0.037, respectively). Stromal localization of TIMP-3 protein was found to have no clinicopathological or prognostic value.ConclusionThis is the first immunohistochemical study to show that TIMP-3 protein within cancer cells is associated with tumor phenotype. Reduced expression of TIMP-3 protein within cancer cells was found to correlate with an aggressive tumor phenotype, negatively affecting the disease-free survival of both subgroups of lymph node-positive and mutant-p53-negative patients.

Does a Single Application of PRP Alter the Expression of IGF-I in the Early Phase of Tendon Healing?

The purpose of this study was to determine whether or not a single application of platelet-rich plasma (PRP) in a ruptured tendon alters the expression of IGF-I in the early phase of healing in an animal wound model. We performed an Achilles tendon rupture model on 48 New Zealand white rabbits, by transecting the tendon transversely and then injecting 0.5 mL of PRP into the tendon mass on one side, and injecting saline on the contralateral, control side. Twenty-four animals received PRP (PRP group), and 24 animals served as untreated controls (control group). Six animals (12 limbs) were killed from each group at 1, 2, 3, and 4 weeks postoperatively. After the animals were killed, 6 paraffin sections were made from each Achilles tendon, 3 of which were stained with hematoxylin and eosin and subjected to microscopic examination, and 3 of which were immunostained with an anti-IGF-I primary antibody. Density of brown diaminobenzidine (DAB) staining was evaluated to quantitatively analyze the results. IGF-I was expressed intracellularly in various cell types throughout the entire healing phase. The growth factor was localized in the epitenon and the endotenon, with an overexpression in the epitenon in the PRP group by the fourth week in comparison with the controls. Furthermore, the tendons treated with PRP healed more rapidly. Based on these findings, PRP could be useful to surgeons treating ruptured tendon.

Application of PRP gel alone or in combination with guided bone regeneration does not enhance bone healing process: An experimental study in rabbits

INTRODUCTION The aim of this study was to assess the hypothesis that application of platelet-rich plasma (PRP) gel in mandibular defects in rabbits, alone or in combination with guided bone regeneration (GBR) techniques, could enhance the bone healing process. MATERIALS AND METHODS Thirty New Zealand white rabbits were used. Three groups of 10 animals each were assigned, and the animals were sacrificed after 12 weeks. During the operation, a rotating trephine bur was used to create circular defects 10-mm in diameter in the region anterior to the jaw angles. In group human fascia lata (HFL), a human fascia lata membrane was used. In group PRP, PRP gel was used to fill the defect, and in group HFL+PRP, PRP was used to fill the defect which after that was covered with a human fascia lata membrane. RESULTS In general, none of the control sides and the PRP treated sides had full development of bone or filling of the defect through bone bridging. Conversely, the sides on which the fascia lata membrane or the combination of membrane and PRP had been applied were characterized mostly by development of newly formed bone that bridged the gap. CONCLUSIONS Our results suggest that the application of PRP gel alone or in combination with GBR does not enhance bone healing process.

Pharmacokinetics of intravitreal bevacizumab (Avastin®) in rabbits.

Purpose: To describe the pharmacokinetics of intravitreal bevacizumab (Avastin®) in rabbits. Methods: The right eye of 20 rabbits was injected intravitreally with 1.25 mg/0.05 mL bevacizumab. Both eyes of four rabbits each time were enucleated at days 1, 3, 8, 15, and 29. Bevacizumab concentrations were measured in serum, aqueous humor, and vitreous. Results: Maximum vitreous (406.25 μg/mL) and aqueous humor (5.83 μg/mL) concentrations of bevacizumab in the right eye were measured at day 1. Serum bevacizumab concentration peaked at day 8 (0.413 μg/mL) and declined to 0.032 μg/mL at 4 weeks. Half-life values in right vitreous, right aqueous humor, and serum were 6.61, 6.51, and 5.87 days, respectively. Concentration of bevacizumab in the vitreous of the noninjected eye peaked at day 8 (0.335 ng/mL) and declined to 0.218 ng/mL at 4 weeks. In the aqueous humor of the noninjected eye, maximum concentration of bevacizumab was achieved at day 8 (1.6125 ng/mL) and declined (to 0.11 ng/mL) at 4 weeks. Conclusion: The vitreous half-life of 1.25 mg/0.05 mL intravitreal bevacizumab was 6.61 days in this rabbit model. Maximum concentrations of bevacizumab were reached at day 1 in both vitreous and aqueous humor of the right eye and at day 8 in the serum. Very low concentrations of bevacizumab were measured in the fellow noninjected eye.

Estrogen anti-inflammatory activity on human monocytes is mediated through cross-talk between estrogen receptor ERα36 and GPR30/GPER1

Estrogens are known modulators of monocyte/macrophage functions; however, the underlying mechanism has not been clearly defined. Recently, a number of estrogen receptor molecules and splice variants were identified that exert different and sometimes opposing actions. We assessed the expression of estrogen receptors and explored their role in mediating estrogenic anti‐inflammatory effects on human primary monocytes. We report that the only estrogen receptors expressed are estrogen receptor‐α 36‐kDa splice variant and G‐protein coupled receptor 30/G‐protein estrogen receptor 1, in a sex‐independent manner. 17‐β‐Estradiol inhibits the LPS‐induced IL‐6 inflammatory response, resulting in inhibition of NF‐κB transcriptional activity. This is achieved via a direct physical interaction of ligand‐activated estrogen receptor‐α 36‐kDa splice variant with the p65 component of NF‐κB in the nucleus. G‐protein coupled receptor 30/G‐protein estrogen receptor 1, which also physically interacts with estrogen receptor‐α 36‐kDa splice variant, acts a coregulator in this process, because its inhibition blocks the effect of estrogens on IL‐6 expression. However, its activation does not mimic the effect of estrogens, on neither IL‐6 nor NF‐κB activity. Finally, we show that the estrogen receptor profile observed in monocytes is not modified during their differentiation to macrophages or dendritic cells in vitro and is shared in vivo by macrophages present in atherosclerotic plaques. These results position estrogen receptor‐α 36‐kDa splice variant and G‐protein coupled receptor 30 as important players and potential therapeutic targets in monocyte/macrophage‐dependent inflammatory processes.

pathologic, radiographic and molecular findings in three fetuses diagnosed with Hem/greenberg skeletal dysplasia

Greenberg skeletal dysplasia is a very rare, autosomal recessive, in utero, lethal chondrodystrophy for which only eight index cases of diverse ethnic origin have been reported so far. The defect is associated with a defect in cholesterol biosynthesis and due to mutations in the gene encoding the lamin B receptor (LBR).

Temporal and spatial expression of TGF-β1 in an Achilles tendon section model after application of platelet-rich plasma

BACKGROUND To investigate the effect of platelet-rich plasma (PRP) on TGF-beta1 expression during tendon healing. METHODS We used 48 skeletally mature New Zealand White rabbits. 24 rabbits received the PRP, and 24 rabbits served as an untreated control group. Equal numbers of animals were sacrificed at 1st, 2nd, 3rd, and 4th week. The surgical procedure involved a transverse incision to transect the Achilles tendon. A volume of 1ml of PRP was then injected into the tendon mass in the PRP group. Histological and immunohistochemical evaluations with an anti-TGF-beta primary antibody were performed. RESULTS The pattern of expression of TGF-beta1 in the PRP group was characterized by a significant upregulation during the first 2 weeks and subsequently significant downregulation in the 3rd and 4th week in comparison with the controls. CONCLUSIONS Our results suggest that PRP may affect the tendon healing process by altering the expression of TGF-beta1.