Josefa P. García-Ruiz

Prolactin is a component of the human synovial liquid and modulates the growth and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

The hormone prolactin (PRL) is the product of a single gene synthesized by pituitary and many extrapituitary tissues. In this study, we have purified and sequenced by mass spectrometry a 29 kDa protein from human synovial liquid, bound to the proteoglycan component of synovial liquid that showed an identical sequence in 20 amino acids to hPRL. We have also found PRL receptor (PRLR) in human knee tissues. The cartilage from osteoarthritic patients shows transcripts of the long PRLR isoform while synovial tissue expresses the intermediate PRLR isoform. Pluripotent mesenchymal stem cells (MSCs) can be isolated from adult bone marrow providing an excellent tool to study MSC-derived differentiation processes. We analyzed the expression of the PRL-PRLR system in hMSCs and during the acquisition of chondrocyte phenotype. We show by RT-PCR that intermediate PRLR isoform is expressed in hMSCs and that PRL exerts a significant increase in cell proliferation. In MSC aggregates cultured in chemically defined medium, we found that extrapituitary PRL transcripts are expressed and the receptor switches isoform expression from the intermediate to long isoform. Furthermore, in cell aggregates, PRL induces type II collagen and extrapituitary PRL expression. Histomorphologic analysis of cell aggregates showed that PRL induces the synthesis of proteoglycans and, in combination with glucocorticoids, a tissue structure with cells organized in longitudinal columns. Under the above conditions, electron microscopic observations show that PRL both downregulates the formation of fibrils of type II collagen and induces cell-cell interactions. All the results presented are consistent with a role of the PRL-PRLR system in bone/cartilage formation/repair processes.

Properties of Average Score Distributions of SEQUEST The Probability Ratio Method

High throughput identification of peptides in databases from tandem mass spectrometry data is a key technique in modern proteomics. Common approaches to interpret large scale peptide identification results are based on the statistical analysis of average score distributions, which are constructed from the set of best scores produced by large collections of MS/MS spectra by using searching engines such as SEQUEST. Other approaches calculate individual peptide identification probabilities on the basis of theoretical models or from single-spectrum score distributions constructed by the set of scores produced by each MS/MS spectrum. In this work, we study the mathematical properties of average SEQUEST score distributions by introducing the concept of spectrum quality and expressing these average distributions as compositions of single-spectrum distributions. We predict and demonstrate in the practice that average score distributions are dominated by the quality distribution in the spectra collection, except in the low probability region, where it is possible to predict the dependence of average probability on database size. Our analysis leads to a novel indicator, the probability ratio, which takes optimally into account the statistical information provided by the first and second best scores. The probability ratio is a non-parametric and robust indicator that makes spectra classification according to parameters such as charge state unnecessary and allows a peptide identification performance, on the basis of false discovery rates, that is better than that obtained by other empirical statistical approaches. The probability ratio also compares favorably with statistical probability indicators obtained by the construction of single-spectrum SEQUEST score distributions. These results make the robustness, conceptual simplicity, and ease of automation of the probability ratio algorithm a very attractive alternative to determine peptide identification confidences and error rates in high throughput experiments.

Functional characterization of human mesenchymal stem cells that maintain osteochondral fates

Adult stem cells are essential for tissue renewal, regeneration and repair, and their expansion in defined culture medium is on focus for regenerative medicine and genetic pathologies. The bone marrow has been shown to be very rich is pluripotent mesenchymal stem cells (MSCs) capable of forming bone, cartilage and also may give rise, to neurons and astrocytes in vivo and in vitro. MSCs can be isolated and expanded in culture, but human cells cannot be verified for a cartilage or a bone fate by transfer experiments. Accordingly, here we used different approaches to characterize hMSCs osteoblastic differentiation in vitro. hMSCs grown in culture in the presence of fetal bovine serum (FBS) expressed the bone‐specific transcription factor Runx2/AML3. When cells were incubated in osteoblastic differentiation medium, cells expressed transcripts belonging to the signaling of Indian HH‐PTHrP axis, GLI transcription factors, and bone target genes including osteopontin. The HH pathway proved to be functional since it induced cells to grow. Cells growing or differentiating to osteoblasts presented the Runx2/AML3 transcription factor, its partner CBFB, and Smad2/3 at the nuclei associated with the nuclear matrix. Furthermore, Runx2/AML3 was observed to co‐localize with SC35 to the nuclear intermediary filaments. These data support the notion that hMSCs isolated from human bone are or become bone progenitor cells upon culture. In the absence of FBS and in the presence of insulin or prolactin, cells show cytoskeletal organization and an AP‐1 transcription site activity resembling proliferative osteochondrocytes while cells in the presence of dexamethasone and added prolactin or TGF‐β resembled differentiated osteoblasts. These specific cellular conditions match those observed during endochondral bone formation. J. Cell. Biochem.

Integration of lipid metabolism in the mammary gland and adipose tissue by prolactin during lactation.

Prolactin deficiency, induced by bromocryptine treatment, brought about reciprocal changes in the ability of adipocytes and acini isolated from lactating rats to synthesize lipids. The capacity to synthesize fatty acids and phospholipids decreased in the mammary gland and increased in adipocytes by bromocryptine treatment. In the mammary gland, the maximum potential activity of the pentose shunt as well as the specific activities of the pathway dehydrogenases were significantly reduced by bromocryptine treatment. Simultaneously, adipose tissue increased its lipogenic capacity but neither the maximum potential of the shunt nor the specific activities of the pentose phosphate shunt dehydrogenases were significantly changed with respect to the control lactating rats. Thus, a differential regulatory mechanism(s) of the pentose phosphate shunt activity appears to operate in these two tissues. Adipocytes from lactating rats showed a poor responsiveness to insulin in terms of lipid synthesis from glucose. In contrast, in adipocytes from bromocryptine treated rats insulin was able to increase lipid synthesis (105%). Sheep prolactin administration ‘in vivo’ partially reversed the effects of bromocryptine. These data suggest that prolactin mediates adipocytes resistance to insulin during lactation. Phospholipid synthesis, as occurred in fatty acid synthesis, is increased in adipose tissue and decreased in mammary gland by bromocryptine treatment. However, α-adrenergic stimulation increases phosphatidylinositol turnover to about the same percentages in both mammary gland acini and adipocytes from lactating rats independently of bromocryptine treatment.

Surface micro- and nano-texturing of stainless steel by femtosecond laser for the control of cell migration

The precise control over the interaction between cells and the surface of materials plays a crucial role in optimizing the integration of implanted biomaterials. In this regard, material surface with controlled topographic features at the micro- and nano-scales has been proved to affect the overall cell behavior and therefore the final osseointegration of implants. Within this context, femtosecond (fs) laser micro/nano machining technology was used in this work to modify the surface structure of stainless steel aiming at controlling cell adhesion and migration. The experimental results show that cells tend to attach and preferentially align to the laser-induced nanopatterns oriented in a specific direction. Accordingly, the laser-based fabrication method here described constitutes a simple, clean, and scalable technique which allows a precise control of the surface nano-patterning process and, subsequently, enables the control of cell adhesion, migration, and polarization. Moreover, since our surface-patterning approach does not involve any chemical treatments and is performed in a single step process, it could in principle be applied to most metallic materials.

Prolactin's role in the early stages of liver regeneration in rats†‡

Liver regeneration after partial hepatectomy (PHx) is a complex process that is regulated by hemodynamic changes, the modulation of cytokines and growth factors, and the activation of immediate early transcription factors that lead to a round of hepatocyte mitosis. Among the factors involved, the pituitary hormone prolactin (PRL) has been shown to induce a hepatotrophic response after partial hepatectomy similar to that caused by phorbol esters; and in isolated hepatocytes PRL triggers a mitogenic response. However, it is becoming clear that PRL exerts a dual role acting in proliferation and differentiation processes. In this work, we have assessed the role of PRL in the early stages of liver regeneration in rats. To this end, three groups of rats were compared: Sham operated, regenerant and regenerant with PRL i.p. administration. Results show that PRL administration prior to partial hepatectomy caused an increase in the binding activity of several transcription factors involved in cell proliferation: AP‐1, c‐Jun and STAT‐3, and in liver‐specific differentiation and maintenance of energetic metabolism: CEBPα, HNF‐1, HNF‐4 at early time points and at later time points HNF‐3. Hepatic sections show that PRL administration increases the number of proliferating cells within 5 h post‐partial hepatectomy. The mRNA of the angiogenic and survival factors VEGF and HIF‐1α, was also induced by PRL treatment. Data indicate that PRL triggers, either directly or indirectly, an acceleration of liver regeneration, preserving liver function and fulfilling a hepatoprotective role. J. Cell. Physiol. 219: 626–633, 2009.

Free-Form Rapid Prototyped Porous PDMS Scaffolds Incorporating Growth Factors Promote Chondrogenesis

In this study, we present a promising approach for the rapid development of porous polydimethylsiloxane (PDMS) scaffold prototypes, with outer geometry defined from the design stage, according to the form of conventional implants or adapted to patients’ biostructures. The manufacture method is based on phase separation processes using materials obtained by casting within additive rapid prototyped molds. We include a comparative study of PDMS sponges obtained by different simple processes. Final in vitro assessment is carried out using hMSCs (bone marrow-derived human mesenchymal stem cells), cultured onto porous PDMS scaffolds functionalized with aminopropyltriethoxysilane (APTS) and equilibrated with a trophic factors medium produced by the cells. Results show that porous PDMS scaffold prototypes are excellent 3D platforms for hMSCs adhesion. Furthermore, this PDMS-3D niche, seeded with hMSCs and chondrogenic incubation medium during three weeks, showed a successful chondrogenesis determined by collagen type II expression. Thus, results show a versatile method to produce a 3D niche to address questions about cartilage and endochondral bone formation or skeleton tissues clinical approaches.

Engineering of silicon surfaces at the micro- and nanoscales for cell adhesion and migration control

The engineering of surface patterns is a powerful tool for analyzing cellular communication factors involved in the processes of adhesion, migration, and expansion, which can have a notable impact on therapeutic applications including tissue engineering. In this regard, the main objective of this research was to fabricate patterned and textured surfaces at micron- and nanoscale levels, respectively, with very different chemical and topographic characteristics to control cell–substrate interactions. For this task, one-dimensional (1-D) and two-dimensional (2-D) patterns combining silicon and nanostructured porous silicon were engineered by ion beam irradiation and subsequent electrochemical etch. The experimental results show that under the influence of chemical and morphological stimuli, human mesenchymal stem cells polarize and move directionally toward or away from the particular stimulus. Furthermore, a computational model was developed aiming at understanding cell behavior by reproducing the surface distribution and migration of human mesenchymal stem cells observed experimentally.

Hybrid luminescent/magnetic nanostructured porous silicon particles for biomedical applications

This work describes a novel process for the fabrication of hybrid nanostructured particles showing intense tunable photoluminescence and a simultaneous ferromagnetic behavior. The fabrication process involves the synthesis of nanostructured porous silicon (NPSi) by chemical anodization of crystalline silicon and subsequent in pore growth of Co nanoparticles by electrochemically-assisted infiltration. Final particles are obtained by subsequent sonication of the Co-infiltrated NPSi layers and conjugation with poly(ethylene glycol) aiming at enhancing their hydrophilic character. These particles respond to magnetic fields, emit light in the visible when excited in the UV range, and internalize into human mesenchymal stem cells with no apoptosis induction. Furthermore, cytotoxicity in in-vitro systems confirms their biocompatibility and the viability of the cells after incorporation of the particles. The hybrid nanostructured particles might represent powerful research tools as cellular trackers or in cellular therapy since they allow combining two or more properties into a single particle.

Effect of prolactin and glucocorticoids on P-enolpyruvate carboxykinase activity in liver and mammary gland from diabetic and lactating rats

SummaryThe administration of 2 bromo-α-ergocryptine, to reduce serum prolactin decreased the activity of cytosolic P-enolpyruvatc carboxykinase (GTP) (EC4.1.1.32) about 50% in both liver and mammary gland of lactating animals. Adrenalectomy had similar effects to those of bromo-a-ergocryptine. In contrast, there was a 50% increase in enzyme activity in the mammary gland of diabetic, lactating rats and a 10-fold increase in liver as compared with normal rats. P-enolpyruvate carboxykinase activity in mammary gland as liver is coordinately regulated by prolactin, glucocorticoids and insulin.