Modification of collagen-based sponges can induce an upshift of the early inflammatory response and a chronic inflammatory reaction led by M1 macrophages: an in vivo study.

BACKGROUND: The present study evaluated the cellular tissue reaction of two equine-derived collagen hemostatic sponges (E-CHS), which differed in thickness after pressing, over 30 days in vivo. The inflammatory response during physiological wound healing in sham-operated animals was used as control group. MATERIAL AND METHODS: First, the E-CHS was pressed by applying constant pressure (6.47 ± 0.85 N) for 2 min using a sterile stainless-steel cylinder until the material was uniformly flattened. Consequently, the original (E-CHS), the pressed (P-E-CHS), as well as the control group (CG; sham operation) were studied independently. The 3 groups were evaluated in vivo after subcutaneous implantation in Wistar rats during 3, 15, and 30 days. Histochemical and immunohistochemical methods provided observations of biomaterial degradation rate, cellular inflammatory response, and vascularization pattern. A derivative of human blood known as platelet-rich fibrin (PRF) was used as an ex vivo model to simulate the initial biomaterial-cell interaction. Segments of E-CHS and P-E-CHS were cultivated for 3 and 6 days with PRF, and the release of pro-inflammatory proteins was measured using ELISA. PRF cultivated alone was used as a control group. RESULTS: At day 3, the CG induced a statistically significant higher presence of monocytes/macrophages (CD68+), pro-inflammatory macrophages (M1; CCR7+), and pro-wound healing macrophages (M2; CD206+) compared to E-CHS and P-E-CHS. At the same time point, P-E-CHS induced a statistically significant higher presence of CD68+ cells compared to E-CHS. After 15 days, E-CHS was invaded by cells and vessels and showed a faster disintegration rate compared to P-E-CHS. On the contrary, cells and vessels were located only in the outer region of P-E-CHS and the biomaterial did not lose its structure and accordingly did not undergo disintegration. The experimental groups induced similar inflammatory reaction primarily with positive pro-inflammatory CD68+/CCR7+ macrophages and a low presence of multinucleated giant cells (MNGCs). At this time point, significantly lower CD68+/CCR7+ macrophages and no MNGCs were detected within the CG when compared to the experimental groups (P < 0.05). After 30 days, E-CHS and P-E-CHS were fully degraded. All groups showed similar inflammatory reaction shifted to a higher presence CD206+ macrophages. A low number of CCR7+ MNGCs were still observable in the implantation bed of both experimental groups. In the ex vivo model, the cells and fibrin from PRF penetrated E-CHS. However, in the case of P-E-CHS, the cells and fibrin stayed on the surface and did not penetrate towards materials central regions. The cultivation of P-E-CHS with PRF induced a statically significant higher release of pro-inflammatory proteins compared to the CG and E-CHS after 3 days. CONCLUSION: Altering the original presentation of a hemostatic sponge biomaterial by pressing modified the initial biomaterial-cell interaction, delayed the early biomaterial's degradation rate, and altered the vascularization pattern. A pressed biomaterial seems to induce a higher inflammatory reaction at early time points. However, altering the biomaterial did not modify the polarization pattern of macrophages compared to physiologic wound healing. The ex vivo model using PRF was shown to be an effective model to simulate the initial biomaterial-cell interaction in vivo. CLINICAL RELEVANCE: A pressed hemostatic sponge could be applied for guided tissue regeneration and guided bone regeneration. In that sense, within the limitations of this study, the results show that the same biomaterial may have two specific clinical indications.

Platelet-rich fibrin secretome induces three dimensional angiogenic activation in vitro.

Different tissue engineering techniques are used to support rapid vascularisation. A novel technique is the use of platelet-rich fibrin (PRF), an autologous source of growth factors. This study was the first to investigate the influence of PRF matrices, isolated following different centrifugation protocols, on human dermal vascular endothelial cells (ECs) in mono-culture and co-culture with human primary fibroblasts (HFs) as an in vitro model for tissue regeneration. Focus was placed on vascular structure formation and growth factor release. HFs and ECs were cultivated with PRF prepared using a high (710 ×g) or low (44 ×g) relative centrifugation force (RCF) over 14 d. Immunofluorescence staining and immunohistochemistry were used to evaluate the microvascular formation. Cell culture supernatants were collected for evaluation of growth factor release. The results showed a PRF-mediated effect on the induction of angiogenesis in ECs. Microvessel-like structure formation was promoted when ECs were combined with low-RCF PRF as compared to high-RCF PRF or control group. The percentage of vascular lumen area was significantly higher in low-RCF PRF, especially at day 7, which coincided with statistically significantly higher growth factor [vascular endothelial factor (VEGF), transforming growth factor ß1 (TGF-ß1) and platelet derived growth factor (PDGF)] concentration measured in low-RCF PRF as compared to high-RCF PRF or control group. In conclusion, reducing the RCF according to the low-speed centrifugation concept (LSCC) resulted in increased growth factor release and angiogenic structure formation with EC mono-culture, suggesting that PRF may be a highly beneficial therapeutic tool for tissue engineering applications.

Modern lipostructure: the use of platelet rich fibrin (PRF).

OBJECTIVES: To evaluate the interest of growth factors adjonction during lipostructure. MATERIALS AND METHODS: Between May 2005 and June 2012, 232 patients benefited from this modern approach of lipostructure with concentrate of platelets. The technique evolves on the simplification way. RESULTS: All the patients were satisfied with the result with minimal associated resorptions. No massive resorption requiring a resumption of lipostructure was noted. CONCLUSION: By offering a matricial support to angiogenesis and by stimulating the proliferation of pre-adipocytes, the PRF could have a beneficial role on the cicatrization and the consolidation of an adipocyte graft.

Reduction of relative centrifugal forces increases growth factor release within solid platelet-rich-fibrin (PRF)-based matrices: a proof of concept of LSCC (low speed centrifugation concept).

Purpose The present study evaluated the platelet distribution pattern and growth factor release (VEGF, TGF-ß1 and EGF) within three PRF (platelet-rich-fibrin) matrices (PRF, A-PRF and A-PRF+) that were prepared using different relative centrifugation forces (RCF) and centrifugation times. Materials and methods immunohistochemistry was conducted to assess the platelet distribution pattern within three PRF matrices. The growth factor release was measured over 10 days using ELISA. Results The VEGF protein content showed the highest release on day 7; A-PRF+ showed a significantly higher rate than A-PRF and PRF. The accumulated release on day 10 was significantly higher in A-PRF+ compared with A-PRF and PRF. TGF-ß1 release in A-PRF and A-PRF+ showed significantly higher values on days 7 and 10 compared with PRF. EGF release revealed a maximum at 24 h in all groups. Toward the end of the study, A-PRF+ demonstrated significantly higher EGF release than PRF. The accumulated growth factor releases of TGF-ß1 and EGF on day 10 were significantly higher in A-PRF+ and A-PRF than in PRF. Moreover, platelets were located homogenously throughout the matrix in the A-PRF and A-PRF+ groups, whereas platelets in PRF were primarily observed within the lower portion. ​Discussion the present results show an increase growthfactor release by decreased RCF. However, further studies must be conducted to examine the extent to which enhancing the amount and the rate of released growth factors influence wound healing and biomaterial-based tissue regeneration. ​Conclusion These outcomes accentuate the fact that with a reduction of RCF according to the previously LSCC (described low speed centrifugation concept), growth factor release can be increased in leukocytes and platelets within the solid PRF matrices.

Reduction of relative centrifugation force within injectable platelet-rich-fibrin (PRF) concentrates advances patients' own inflammatory cells, platelets and growth factors: the first introduction to the low speed centrifugation concept.

PURPOSE: The aim of this study was to analyze systematically the influence of the relative centrifugation force (RCF) on leukocytes, platelets and growth factor release within fluid platelet-rich fibrin matrices (PRF). MATERIALS AND METHODS: Systematically using peripheral blood from six healthy volunteers, the RCF was reduced four times for each of the three experimental protocols (I-III) within the spectrum (710-44 g), while maintaining a constant centrifugation time. Flow cytometry was applied to determine the platelets and leukocyte number. The growth factor concentration was quantified 1 and 24 h after clotting using ELISA. RESULTS: Reducing RCF in accordance with protocol-II (177 g) led to a significantly higher platelets and leukocytes numbers compared to protocol-I (710 g). Protocol-III (44 g) showed a highly significant increase of leukocytes and platelets number in comparison to -I and -II. The growth factors' concentration of VEGF and TGF-ß1 was significantly higher in protocol-II compared to -I, whereas protocol-III exhibited significantly higher growth factor concentration compared to protocols-I and -II. These findings were observed among 1 and 24 h after clotting, as well as the accumulated growth factor concentration over 24 h. DISCUSSION: Based on the results, it has been demonstrated that it is possible to enrich PRF-based fluid matrices with leukocytes, platelets and growth factors by means of a single alteration of the centrifugation settings within the clinical routine. CONCLUSIONS: We postulate that the so-called low speed centrifugation concept (LSCC) selectively enriches leukocytes, platelets and growth factors within fluid PRF-based matrices. Further studies are needed to evaluate the effect of cell and growth factor enrichment on wound healing and tissue regeneration while comparing blood concentrates gained by high and low RCF.

[Influence of platelet rich fibrin (PRF) on proliferation of human preadipocytes and tympanic keratinocytes: A new opportunity in facial lipostructure (Coleman's technique) and tympanoplasty?]. / De l'influence du platelet rich fibrin (PRF) sur la prolifération des préadipocytes et keratinocytes tympaniques humains: une nouvelle opportunité pour la lipostructure faciale (technique de Coleman) et la tympanoplastie?

AIM: To analyze the effects of PRF (a platelet and immune autologous concentrate) on in vitro proliferation of human keratinocytes and preadipocytes, and to determine if these results may offer an opening on new clinical investigations, particularly in the improvement of tympanoplasties and facial lipostructures (Coleman's technique). MATERIALS AND METHODS: Human tympanic keratinocytes and preadipocytes are collected and cultured using the explant technique. 4 series of each type of cells are cultivated either in normal condition (control group) or with PRF (test group). The Petri dishes (of culture) are taken out on the 3rd, 7th, 14th and 21st day, for counting. Evolutions of cells' number are analyzed with a variance test. RESULTS: The number of cells in culture increases of more than 60% on the 7th day, and of almost 150% right from the 14th day when in presence of PRF. The daily proliferation peak occurs around the 14th day. The two cellular tested types react similarly. CONCLUSION: The PRF, considered as a healing biomaterial, could be used in tympanic and facial lipostructures surgeries, in order to improve the therapeutic result. Other applications in microsurgery and in plastic surgery may be possible, but specific clinical studies need to validate such protocols.

Recurrent nonimmune hydrops fetalis: a rare presentation of sialic acid storage disease.

A case of recurrent hydrops fetalis, diagnosed on second trimester's ultrasonography, has led to the diagnosis of sialic acid storage disease. No classic etiology was found after the first accident. The recurrence in subsequent pregnancy raised the possibility of a storage disease that was confirmed by amniocentesis. The diagnosis of Salla's disease was based on high levels of free sialic acid in amniotic fluid and fetal cells culture and by specific histologic features on fetopathologic examination. Diagnosis of inherited diseases is important because it implies a high risk of recurrence which makes mandatory genetic counseling and prenatal care in subsequent pregnancies.

[In utero study of the eye of normal fetuses. Static ultrasonographic aspects and clinical implications]. / Etude in utero de l'oeil du foetus normal. Aspects échographiques statiques et implications cliniques.

PURPOSE OF THE STUDY: Some ocular diseases are detected by amniotic fluid analysis. However, some serious eye malformations are only detected at birth. By analogy to other organs, we were concerned by fetal sonography studying in utero ocular structures. METHODS: We performed 150 fetal sonograph with 2 abdominal probes and one vaginal probe. We defined: (a) Axes where ocular structures are best visualised, (b) sonographic images of these structures, (c) the date at which these structures are detected. RESULTS: Orbit are detected between 11th and 12th week amenorrhae. Lens are detected between 12th and 14th week. Hyaloid artery appears around the 18th and disappears around the 32nd. Lids were recognised at 16th week. These results were confirmed by similar studies. Pathologic cases described in the literature are discussed. CONCLUSION: Fetal sonography must include a precise study of the eye. However, the eye is a small organ, so its study implicates technically skilled and an experienced practitioner. Fetal sonography gives precise information about normal eye development and allows the detection of structural anomalies (anophthalmos, microphthalmos, cyclopia) or orbital prenatal malformations (hyaloid artery persistance).

[Simultaneous intra- and extra- uterine pregnancies (author's transl)]. / Grossesses intra-utérines et extra-utérines simultanées.

Six further cases of simultaneous intra- and extra-uterine pregnancies are reported. The findings confirm those reported in the published literature. The frequency of such cases in one in 30 000 pregnancies. It is evident, however, that some cases, in which spontaneous abortion occurs, remain unrecognized, the haemorrhage being attributed to the extra-uterine gestation. In 90 p. cent of cases they are detected because of the extra-uterine pregnancy, and their clinical course is very variable. The association of pregnancies is almost as frequent as triplets but is very rarely mentioned. Valid clinical and pathogenic data could be obtained from their more systematic study.

[Triploidy and mole. Apropos of 2 cases continuing to the second trimester]. / Triploïdie et môle. A propos de deux cas évolutifs du deuxième trimestre.

Triploidy in a common chromosomal abnormality that gives rise to early abortion in most cases. Rarely triploidies carry on past the end of the first trimester. When they do they are always accompanied by severe intra-uterine growth retardation with occasionally molar changes in the placenta. These changes are different from hydatidiform moles but they can become malignant. The formal diagnosis of triploidy depends on the fetal caryotype.