Monica Nelea

Meniscus structure in human, sheep, and rabbit for animal models of meniscus repair.

Meniscus injury is a frequently encountered clinical orthopedic issue and is epidemiologically correlated to osteoarthritis. The development of new treatments for meniscus injury is intimately related to the appropriateness of animal models for their investigation. The purpose of this study was to structurally compare human menisci to sheep and rabbit menisci to generate pertinent animal models for meniscus repair. Menisci were analyzed histologically, immunohistochemically, and by environmental scanning electron microscopy (ESEM). In all species, collagen I appeared throughout most menisci, but was absent from the inner portion of the tip in some samples. Collagen II was present throughout the inner main meniscal body, while collagen VI was found in pericellular and perivascular regions. The glycosaminoglycan‐rich inner portion of menisci was greater in area for rabbit and sheep compared to human. Cells were rounded in central regions and more fusiform at the surface, with rabbit being more cellular than sheep and human. Vascular penetration in rabbit was confined to the very outermost region (1% of meniscus length), while vessels penetrated deeper into sheep and human menisci (11–15%). ESEM revealed a lamellar collagenous structure at the articulating surfaces of sheep and human menisci that was absent in rabbit. Taken together, these data suggest that the main structural features that will influence meniscus repair—cellularity, vascularity, collagen structure—are similar in sheep and human but significantly different in rabbit, motivating the development of ovine meniscus repair models.

Chondrocyte Aggregation in Suspension Culture Is GFOGER-GPP- and β1 Integrin-dependent

Isolated chondrocytes form aggregates in suspension culture that maintain chondrocyte phenotype in a physiological pericellular environment. The molecular mechanisms involved in chondrocyte aggregation have not been previously identified. Using this novel suspension culture system, we performed mRNA and protein expression analysis along with immunohistochemistry for potential cell adhesion molecules and extracellular matrix integrin ligands. Inhibition of aggregation assays were performed using specific blocking agents. We found that: (i) direct cell-cell interactions were not involved in chondrocyte aggregation, (ii) chondrocytes in aggregates were surrounded by a matrix rich in collagen II and cartilage oligomeric protein (COMP), (iii) aggregation depends on a β1-integrin, which binds a triple helical GFOGER sequence found in collagens, (iv) integrin α10-subunit is the most highly expressed α-subunit among those tested, including α5, in aggregating chondrocytes. Taken together, this body of evidence suggests that the main molecular interaction involved in aggregation of phenotypically stable chondrocytes is the α10β1-collagen II interaction.

Chitosan-based therapeutic nanoparticles for combination gene therapy and gene silencing of in vitro cell lines relevant to type 2 diabetes

Glucagon like peptide 1 (GLP-1), a blood glucose homeostasis modulating incretin, has been proposed for the treatment of type 2 diabetes mellitus (T2DM). However, native GLP-1 pharmacokinetics reveals low bioavailability due to degradation by the ubiquitous dipeptydil peptidase IV (DPP-IV) endoprotease. In this study, the glucosamine-based polymer chitosan was used as a cationic polymer-based in vitro delivery system for GLP-1, DPP-IV resistant GLP-1 analogues and siRNA targeting DPP-IV mRNA. We found chitosans to form spherical nanocomplexes with these nucleic acids, generating two distinct non-overlapping size ranges of 141-283 nm and 68-129 nm for plasmid and siRNA, respectively. The low molecular weight high DDA chitosan 92-10-5 (degree of deacetylation, molecular weight and N:P ratio (DDA-Mn-N:P)) showed the highest plasmid DNA transfection efficiency in HepG2 and Caco-2 cell lines when compared to 80-10-10 and 80-80-5 chitosans. Recombinant native GLP-1 protein levels in media of transfected cells reached 23 ng/L while our DPP-IV resistant analogues resulted in a fivefold increase of GLP-1 protein levels (115 ng/L) relative to native GLP-1, and equivalent to the Lipofectamine positive control. We also found that all chitosan-DPP-IV siRNA nanocomplexes were capable of DPP-IV silencing, with 92-10-5 being significantly more effective in abrogating enzymatic activity of DPP-IV in media of silenced cells, and with no apparent cytotoxicity. These results indicate that specific chitosan formulations may be effectively used for the delivery of plasmid DNA and siRNA in a combination therapy of type 2 diabetes.

The double capsules in macro-textured breast implants.

Breast implants are amongst the most widely used types of permanent implants in modern medicine and have both aesthetic and reconstructive applications with excellent biocompatibility. The double capsule is a complication associated with textured prostheses that leads to implant displacement; however, its etiology has yet to be elucidated. In this study, 10 double capsules were sampled from breast expander implants for in-depth analysis; histologically, the inner capsular layer demonstrated highly organized collagen in sheets with delamination of fibers. At the prosthesis interface (PI) where the implant shell contacts the inner capsular layer, scanning electron microscopy (SEM) revealed a thin layer which mirrored the three-dimensional characteristics of the implant texture; the external surface of the inner capsular layer facing the intercapsular space (ICS) was flat. SEM examination of the inner capsule layer revealed both a large bacterial presence as well as biofilm deposition at the PI; a significantly lower quantity of bacteria and biofilm were found at the ICS interface. These findings suggest that the double capsule phenomenons etiopathogenesis is of mechanical origin. Delamination of the periprosthetic capsule leads to the creation of the ICS; the maintained separation of the 2 layers subsequently alters the biostability of the macro-textured breast implant.

Injectable chitosan‐platelet‐rich plasma (PRP) implants to promote tissue regeneration: In vitro properties, in vivo residence, degradation, cell recruitment and vascularization

The purpose of this study was to develop freeze‐dried chitosan formulations that can be solubilized in platelet‐rich plasma (PRP) to form injectable implants for tissue repair. A systematic approach to adjust formulation parameters, including chitosan number average molar mass (Mn), chitosan concentration and lyoprotectant concentration, was undertaken to identify compositions that would rapidly (< 1 min) and completely solubilize in PRP, would have paste‐like handling properties upon solubilization and coagulate rapidly (< 5 min) to form solid chitosan‐PRP hybrid implants that are stable and homogenous. Freeze‐dried cakes containing calcium chloride, as well as distinct chitosan Mn, chitosan concentration and lyoprotectant concentration, were prepared. PRP was used to solubilize the freeze‐dried cakes and assess in vitro and in vivo performance, the latter as dorsal subcutaneous injections into New Zealand White rabbits. Freeze‐dried polymer formulations containing low and medium chitosan Mn and concentrations were rapidly and completely solubilized in PRP. The paste‐like chitosan‐PRP mixtures coagulated quickly to form solid chitosan‐PRP hybrids, which retracted much less than PRP‐only controls. Homogeneous dispersion of chitosan within the hybrid clots was strongly dependent on chitosan Mn, and occurred only with medium Mn chitosan. Chitosan‐PRP hybrid clots were resident subcutaneously in vivo until at least 2 weeks while PRP controls were quickly degraded in one day. Compared to PRP alone, chitosan‐PRP hybrids had much greater capacity to induce local cell recruitment accompanied by angiogenesis, suggesting a strong potential for their use in regenerative medicine. Copyright

Preparation of Concentrated Chitosan/DNA Nanoparticle Formulations by Lyophilization for Gene Delivery at Clinically Relevant Dosages

Chitosan/DNA polyplexes have been optimized for efficient and safe in vitro and in vivo gene delivery. Clinical application of this technology requires the development of formulations with higher concentrations to reach therapeutic dosages. Polyplexes were prepared using chitosan and EGFPLuc plasmids. Freeze-thawing and freeze-drying studies were performed to identify and optimize lyoprotectant and buffer contents in formulations. Freeze-dried samples were rehydrated in reduced volumes to increase their final DNA dose. Nanoparticle physicochemical properties were analyzed, and their transfection efficiency and cytotoxicity were measured in human embryonic kidney 293 cells. Data showed that 3.5 mM histidine buffer (pH 6.5) combined with one of 0.5% wt/vol sucrose, dextran 5 kDa, or trehalose was required to prevent polyplex aggregation during freeze-drying. Optimal formulations could be concentrated 20-fold, to a clinically desired ∼1 mg of DNA/mL, while maintaining near physiological pH and tonicity. Polyplexes were predominantly spherical, with diameters below 200 nm, polydispersity indexes below 0.32, and zeta potentials above +19 mV. Rehydrated formulations had transfection efficiencies no less than 65% of fresh polyplexes without excipients and had no effect on viability and metabolic activity of human embryonic kidney 293 cells. These concentrated formulations represent an important step toward clinical use of chitosan-based gene delivery systems.

Chitosan inhibits platelet-mediated clot retraction, increases platelet-derived growth factor release, and increases residence time and bioactivity of platelet-rich plasma in vivo

Platelet-rich plasma (PRP) has been used to treat different orthopaedic conditions, however, the clinical benefits of using PRP remain uncertain. Chitosan (CS)-PRP implants have been shown to improve meniscus, rotator cuff and cartilage repair in pre-clinical models. The purpose of this current study was to investigate in vitro and in vivo mechanisms of action of CS-PRP implants. Freeze-dried formulations containing 1% (w/v) chitosan (80% degree of deacetylation and number average molar mass 38 kDa), 1% (w/v) trehalose as lyoprotectant and 42.2 mM calcium chloride as clot activator were solubilized in PRP. Gravimetric measurements and molecular/cellular imaging studies revealed that clot retraction is inhibited in CS-PRP hybrid clots through physical coating of platelets, blood cells and fibrin strands by chitosan, which interferes with platelet aggregation and platelet-mediated clot retraction. Flow cytometry and ELISA assays revealed that platelets are activated and granules secreted in CS-PRP hybrid clots and that cumulative release of PDGF-AB and EGF is higher from CS-PRP hybrid clots compared to PRP clots in vitro. Finally, CS-PRP implants resided for up to 6 weeks in a subcutaneous implantation model and induced cell recruitment and granulation tissue synthesis, confirming greater residency and bioactivity compared to PRP in vivo.

Delamination Is a Major Etiologic Factor of Double Capsules Around Textured Implants.

Methods: Using scanning electronic microscopy (SEM) and histology we analyzed 10 patients presenting double capsule phenomenon around textured Allergan Biocell® implants, for a total of 20 samples: the inner capsule along with the prosthesis shell and the outer capsule. The inner capsule was analyzed at both the prosthesis interface (PI) and the outer surface in contact with inter-capsular space (ICS). We evaluated bacterial density and biofilm using the Van Heerden scale on both surfaces of the inner. We correlated the bacterial density to implant age.