Thierry Balaguer

Human Primary Osteocyte Differentiation in a 3D Culture System

Studies on primary osteocytes, which compose >90–95% of bone cells, embedded throughout the mineralized matrix, are a major challenge because of their difficult accessibility and the very rare models available in vitro. We engineered a 3D culture method of primary human osteoblast differentiation into osteocytes. These 3D‐differentiated osteocytes were compared with 2D‐cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for 4 wk in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times, as well as by quantitative RT‐PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Histological analysis showed, after only 1 wk, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte‐like cells. Real‐time PCR expression of a set of bone‐related genes confirmed their osteocyte phenotype. Comparison with plastic‐cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid‐osteocytes. This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast‐osteocyte differentiation pathway, the osteocyte interaction with the other bone cells, and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the cross‐talk of osteocytes with tumor cells in bone metastasis, because it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could also be a very efficient tool for drug testing and bone tissue engineering applications.

Onychomatricoma: New Clinical and Histological Features. A Review of 19 Tumors

To further define the clinicopathological spectrum of onychomatricoma (OM). We report the clinical feature, histological, and immunophenotypic characteristics of 19 cases of OM diagnosed between 2002 and 2007. The characteristic histologic appearance of OM is sometimes difficult to grasp because of 3 main factors: the anatomic particularities of the nail apparatus, the often fragmented aspect of the tissue specimen, and the choice of the section planes, which strongly modified the morphologic appearances observed. To prevent these difficulties, we built a tridimensional model using serial, transverse, and longitudinal sections. This reconstitution gives us a better understanding of the apparent diversity of the morphologic aspects observed in linking them to the anatomic site of the tumor. OM of the matrix is characterized by a thick nail plate with porch roof. OM of the ventral aspect of the proximal nail fold (PNF) is characterized by a nail plate without porch roof, exhibiting either a woodworm-like appearance or multiple cavities. In this second category, the fibrous base becomes elongated in shape, taking the shape of the anatomic contour of the PNF. The stroma gives rise to numerous fibroepithelial digitations. This pattern is different from the classical OM visualized in longitudinal sections, which appears as a single and large fibroepithelial tumor, that is, the multiple distal epithelial digitations arranged along a transversal plane are not seen. In the PNF variant, the characteristic clinical signs of OM fail to appear. We individualize 3 misleading clinical variants: tumor with a verrucous surface that is located in the lateral nail fold, as a band pattern suggesting wart or Bowen disease; a total dystrophy of the nail unit mimicking a squamous cell carcinoma; and pseudofibrokeratoma type. In the OM located on the ventral matrix, 3 new specific histologic variants were noted: pleomorphic OM, OM with a predominantly collagenous stroma, and superficial acral fibromyxoma-;like OM. OM is a benign tumor with a broader morphologic spectrum than previously thought. When the nail plate is not available, the immunohistochemistry can aid diagnosis by highlighting the peculiar immunophenotyping of OM, which expresses CD34 but not CD99, epithelial membrane antigen, S-100 protein, actin, and desmin.

Onychomatricoma in the light of the microanatomy of the normal nail unit.

Onychomatricoma (OM) is an uncommon benign tumor of the nail thought to exhibit differentiation limited toward the nail matrix. Four recent articles from our laboratory have shown, in some respect, a morphological and immunohistochemical homology between the nail unit and the hair follicle at the level of the matrix and isthmus. The purposes of this article are as follows: to investigate whether the sequential pattern of hair keratin expression in the normal nail matrix is maintained in OM, to compare and contrast follicular tumors with matrix differentiation in OM, and to furnish morphological and immunohistochemical markers of the onychogenic capacity of OM. Formalin-fixed paraffin sections from 6 OM were examined using specific keratin (K) antibodies for the matrix, nail bed, and nail isthmus. Hair keratins were expressed in a sequential pattern similar to normal nail matrix. In 3 cases where the cavities were completely lined by the fibroepithelial projections, the morphological aspect and the pattern of expression of K5, K17, K6, K16, and K75 suggested a differentiation toward the nail bed and the nail isthmus. This study shows for the first time that OM can recapitulate the entire nail unit with differentiation toward the nail bed and the nail isthmus. We have identified new histopathological and immunohistochemical features in OM, and we have abridged the diversity of its histological presentation in 2 main patterns: a lobulated or foliated pattern, observed principally on transverse section, and a “glove-finger” mono- or multidigitate pattern, observed mainly on longitudinal section. We have also concluded that OM is not a nail variant of trichoblastoma, pilomatricoma, or other pilar tumors. The concept of epithelial onychogenic tumor with onychogenic mesenchyme could shed more light about the true nature of this peculiar mixed tumor. However, the term OM is short and sanctioned by usage, which justifies keeping it.

Adaptive immune response inhibits ectopic mature bone formation induced by BMSCs/BCP/plasma composite in immune-competent mice.

A combination of autologous bone marrow stromal cells (BMSCs) and biomaterials is a strategy largely developed in bone tissue engineering, and subcutaneous implantation in rodents or large animals is often a first step to evaluate the potential of new biomaterials. This study aimed at investigating the influence of the immune status of the recipient animal on BMSCs-induced bone formation. BMSCs prepared from C57BL/6 mice, composed of a mixture of mesenchymal stromal and monocytic cells, were combined with a biomaterial that consisted of biphasic calcium phosphate (BCP) particles and plasma clot. This composite was implanted subcutaneously either in syngenic C57BL/6 immune-competent mice or in T-lymphocyte-deficient Nude (Nude) mice. Using histology, immunohistochemistry, and histomorphometry, we show here that this BMSC/BCP/plasma clot composite implanted in Nude mice induces the formation of mature lamellar bone associated to hematopoietic areas and numerous vessels. Comparatively, implantation in C57BL/6 results in the formation of woven bone without hematopoietic tissue, a lower number of new vessels, and numerous multinucleated giant cells (MNGCs). In situ hybridization, which enabled to follow the fate of the BMSCs, revealed that BMSCs implanted in Nude mice survived longer than BMSCs implanted in C57BL/6 mice. Quantitative expression analysis of 280 genes in the implants indicated that the differences between C57BL/6 and Nude implants corresponded almost exclusively to genes related to the immune response. Gene expression profile in C57BL/6 implants was consistent with a mild chronic inflammation reaction characterized by Th1, Th2, and cytotoxic T-lymphocyte activation. In the implants retrieved from T-deficient Nude mice, Mmp14, Il6st, and Tgfbr3 genes were over-expressed, suggesting their putative role in bone regeneration and hematopoiesis. In conclusion, we show here that the T-mediated inflammatory microenvironment is detrimental to BMSCs-induced bone formation and shortens the survival of implanted cells. Conversely, the lack of T-lymphocyte reaction in T-deficient animals is beneficial to BMSCs-induced mature bone formation. This should be taken into account when evaluating cell/biomaterial composites in these models.

Visfatin expression analysis in association with recruitment and activation of human and rodent brown and brite adipocytes

ABSTRACT Human brown adipocytes are able to burn fat and glucose and are now considered as a potential strategy to treat obesity, type 2 diabetes and metabolic disorders. Besides their thermogenic function, brown adipocytes are able to secrete adipokines. One of these is visfatin, a nicotinamide phosphoribosyltransferase involved in nicotinamide dinucleotide synthesis, which is known to participate in the synthesis of insulin by pancreatic β cells. In a therapeutic context, it is of interest to establish whether a potential correlation exists between brown adipocyte activation and/or brite adipocyte recruitment, and adipokine expression. We analyzed visfatin expression, as a pre-requisite to its secretion, in rodent and human biopsies and cell models of brown/brite adipocytes. We found that visfatin was preferentially expressed in mature adipocytes and that this expression was higher in brown adipose tissue of rodents compared to other fat depots. However, using various rodent models we were unable to find any correlation between visfatin expression and brown or brite adipocyte activation or recruitment. Interestingly, the situation is different in humans where visfatin expression was found to be equivalent between white and brown or brite adipocytes in vivo and in vitro. In conclusion, visfatin can be considered only as a rodent brown adipocyte biomarker, independently of tissue activation.

Acquired Localized Longitudinal Pachyonychia and Onychomatrical Tumors: A Comparative Study to Onychomatricomas (5 Cases) and Onychocytic Matricomas (4 Cases).

Background:Besides onychomatricoma (OM), which shows a clinical band pattern of nail plate thickening, 2 new onychomatrical tumors with this clinical feature have recently been described: onychocytic matricoma (OCM) and in situ onychocytic carcinoma. Objective:The purpose of this study was to present 4 cases of OCMs and compare their clinical and histopathologic characteristics with usual OMs. Methods:We studied 4 cases of OCMs with nail clipping in 3 cases and an extensive immunohistochemical study for hair-related keratins and epithelial keratins. Nail clipping of OCMs was compared with the distal nail plate of 5 cases of OMs. Results:All cases showed an acquired localized longitudinal band pattern of a thickened nail plate with yellow discoloration in 2 cases and a black streak in 2 cases. All cases showed a V-shaped keratogenous epithelial tumor with a papillomatous pattern of growth. The nail plate was thickened with small holes in a honeycomb pattern. In contrast, the 5 OMs showed the classical pattern of a panonychoma fibropapilliferum. The nail plate showed large cavities in a honeycomb pattern. Conclusions:This case series raises awareness of the clinical value of longitudinal pachyonychia coupled with nail clipping in the early detection of onychomatrical tumors as generic diagnosis with a limited differential diagnosis and a simple therapeutic approach. Nail clipping could be an aid in the surgical planning of onychomatrical tumor. A diagnosis of a benign growth could be suggested when the average dimensions of cavities are superior to 0.15 mm sparing the patient from an excisional procedure with its risk of subsequent permanent nail dystrophy. In contrast, nail clipping with a honeycomb pattern of minute cavities with average dimension inferior to 0.10 mm should prompt a biopsy of the distal matrix to rule out a malignant lesion.

Calcium supplementation decreases BCP-induced inflammatory processes in blood cells through the NLRP3 inflammasome down-regulation

Interaction of host blood with biomaterials is the first event occurring after implantation in a bone defect. This study aimed at investigating the cellular and molecular consequences arising at the interface between whole blood and biphasic calcium phosphate (BCP) particles. We observed that, due to calcium capture, BCP inhibited blood coagulation, and that this inhibition was reversed by calcium supplementation. Therefore, we studied the impact of calcium supplementation on BCP effects on blood cells. Comparative analysis of BCP and calcium supplemented-BCP (BCP/Ca) effects on blood cells showed that BCP as well as BCP/Ca induced monocyte proliferation, as well as a weak but significant hemolysis. Our data showed for the first time that calcium supplementation of BCP microparticles had anti-inflammatory properties compared to BCP alone that induced an inflammatory response in blood cells. Our results strongly suggest that the anti-inflammatory property of calcium supplemented-BCP results from its down-modulating effect on P2X7R gene expression and its capacity to inhibit ATP/P2X7R interactions, decreasing the NLRP3 inflammasome activation. Considering that monocytes have a vast regenerative potential, and since the excessive inflammation often observed after bone substitutes implantation limits their performance, our results might have great implications in terms of understanding the mechanisms leading to an efficient bone reconstruction. STATEMENT OF SIGNIFICANCE Although scaffolds and biomaterials unavoidably come into direct contact with blood during bone defect filling, whole blood-biomaterials interactions have been poorly explored. By studying in 3D the interactions between biphasic calcium phosphate (BCP) in microparticulate form and blood, we showed for the first time that calcium supplementation of BCP microparticles (BCP/Ca) has anti-inflammatory properties compared to BCP-induced inflammation in whole blood cells and provided information related to the molecular mechanisms involved. The present study also showed that BCP, as well as BCP/Ca particles stimulate monocyte proliferation. As monocytes represent a powerful target for regenerative therapies and as an excessive inflammation limits the performance of biomaterials in bone tissue engineering, our results might have great implications to improve bone reconstruction.