Hydrolat of Helichrysum italicum promotes tissue regeneration during wound healing.

Wound healing is a dynamic process involving different cell types with distinct roles according to the stages of healing. Fibroblasts and stem cells actively participate in tissue regeneration. A proper stimulation could contribute to enhance wound healing process-es. Helichrysum italicum (H. italicum) is a medical plant well described for its pharmacological, antimicrobial, and anti-inflammatory activities. Aim of the present work was to examine the effect of the hydrolat derivate from H. italicum on stem cells isolated from skin and fibroblasts in vitro in presence or absence of tissue damage. The viability and proliferation of all cell types cultured in dif-ferent conditions were analyzed by MTT and BrdU assays. Cell proliferation after wound was analyzed with scratch test. Also, the expression of the main genes involved in tissue repair was evaluated by RT-qPCR analysis. Here we describe the capability of hy-drolat of H. italicum to promote tissue regeneration after scratch test both in stem cells and in fibroblasts. Moreover, the gene ex-pression analysis revealed that, hydrolat of H. italicum is also able to enhance stemness related. In conclusion our results are en-couraging, highlighting novel regenerative properties of hydrolat of H. italicum and paving the way for future application of this wasting product in accelerating wound healing.

Liquid resorbable nanofibrous surgical mesh: a proof of a concept.

BACKGROUND: Surgical mesh is widely used not only to treat but also to prevent incisional hernia formation. Despite much effort by material engineers, the 'ideal' mesh mechanically, biologically and surgically easy to use remains elusive. Advances in tissue engineering and nanomedicine have allowed new concepts to be tested with promising results in both small and large animals. Abandoning the concept of a pre-formed mesh completely for a 'pour in liquid mesh' has never been tested before. MATERIALS AND METHODS: Thirty rabbits underwent midline laparotomy with closure using an absorbable suture and small stitch small bites technique. In addition, their abdominal wall closure was reinforced by a liquid nanofibrous scaffold composed of a fibrin sealant and nanofibres of poly-ε-caprolactone with or without hyaluronic acid or the sealant alone, poured in as an 'onlay' over the closed abdominal wall. The animals were killed at 6 weeks and their abdominal wall was subjected to histological and biomechanical evaluations. RESULTS: All the animals survived the study period with no major complication. Histological evaluation showed an eosinophilic infiltration in all groups and foreign body reaction more pronounced in the groups with nanofibres. Biomechanical testing demonstrated that groups treated with nanofibres developed a scar with higher tensile yield strength. CONCLUSION: The use of nanofibres in a liquid form applied to the closed abdominal wall is easy to use and improves the biomechanical properties of healing fascia at 6 weeks after midline laparotomy in a rabbit model.

Dynamic creep properties of a novel nanofiber hernia mesh in abdominal wall repair.

PURPOSE: Incisional hernia is the most common complication following abdominal surgery. While mesh repair is common, none of the current meshes mimic the physiology of the abdominal wall. This study compares suture only repair with polypropylene mesh and a prototype of a novel implant (poly-epsilon-caprolactone nanofibers) and their influence on the physiology of an abdominal wall in an animal model. METHODS: 27 Chinchilla rabbits were divided into six groups based on the type of the implant. Midline abdominal incision was repaired using one of the compared materials with suture alone serving as the control. 6 weeks post-surgery animals were killed and their explanted abdominal wall subjected to biomechanical testing. RESULTS: Both-hysteresis and maximum strength curves showed high elasticity and strength in groups where the novel implant was used. Polypropylene mesh proved as stiff and fragile compared to other groups. CONCLUSION: Poly-epsilon-caprolactone nanofiber scaffold is able to improve the dynamic properties of healing fascia with no loss of maximum tensile strength when compared to polypropylene mesh in an animal model.

Needleless electrospun and centrifugal spun poly-ε-caprolactone scaffolds as a carrier for platelets in tissue engineering applications: A comparative study with hMSCs.

The biofunctionalization of scaffolds for tissue engineering is crucial to improve the results of regenerative therapies. This study compared the effect of platelet-functionalization of 2D electrospun and 3D centrifugal spun scaffolds on the osteogenic potential of hMSCs. Scaffolds prepared from poly-ε-caprolactone, using electrospinning and centrifugal spinning technology, were functionalized using five different concentrations of platelets. Cell proliferation, metabolic activity and osteogenic differentiation were tested using hMSCs cultured in differential and non-differential medium. The porous 3D structure of the centrifugal spun fibers resulted in higher cell proliferation. Furthermore, the functionalization of the scaffolds with platelets resulted in a dose-dependent increase in cell metabolic activity, proliferation and production of an osteogenic marker - alkaline phosphatase. The effect was further promoted by culture in an osteogenic differential medium. The increase in combination of both platelets and osteogenic media shows an improved osteoinduction by platelets in environments rich in inorganic phosphate and ascorbate. Nevertheless, the results of the study showed that the optimal concentration of platelets for induction of hMSC osteogenesis is in the range of 900-3000 × 109 platelets/L. The study determines the potential of electrospun and centrifugal spun fibers with adhered platelets, for use in bone tissue engineering.

PVA and PCL nanofibers are suitable for tissue covering and regeneration.

The aim of the study was to evaluate the safety and efficacy of a new therapeutic approach to skin defects resulting from split thickness grafting. Within the study, nanofiber-based dressings fabricated using polyvinyl alcohol (PVA) and poly-ε-caprolactone (PCL) were used, with different mass density. The study was performed in 1 female minipig. Nine defects (approx. 4x4 cm) were made in the superficial skin layer. The tested materials were applied to the squared skin defect and covered by a Jelonet paraffin gauze, sutured in the corners of the defects. The animal was monitored daily during the healing process (21 days). On day 5, 12, and 27, the healing of the wound was evaluated, and a biopsy was performed for further histologic testing. At the end of the study (on day 27 after the procedure), the animal was euthanized, and a standard pathologic evaluation was performed. We can conclude that the nanofiber scaffold which was well tolerated, could be used as a smart skin cover which could be functionalized with another bioactive substances directly on the surgeon table, among potential bioactive substances belong platelet derivatives, antibiotics, etc.

The dead space after extirpation of rectum. Current management and searching for new materials for filling.

Pelvic surgeries such as extirpation of the rectum or pelvic exenteration lead to a creation of a dead space, which can be cause of complication, such as bowel obstruction, perineal hernia, abscess or hematoma. A growing incidence of complication is expected in connection with the increasing use of laparoscopic and robotic approaches or ELAPE method. Since the bone structures do not allow compression, the only way to deal with the dead space is to fill it in. Present methods provide the filling with omental or myofascial flaps. The length and the mobility of the omental flap can be the limitation. Myofascial flaps are technically more demanding and bring the complications of a donor place. Synthetic or biological meshes do not deal with dead space problematic. Modern technologies using nanomaterials offer the possibility to produce a material with specific properties for example shape, inner structure, surface, or time of degradation. The modified material could also satisfy the requirements for filling the dead space after surgeries.

Electrospun nanofibers as support for the healing of intestinal anastomoses.

The breakdown of intestinal anastomosis is a serious postsurgical complication. The worst complication is anastomotic leakage, resulting in contaminated peritoneal cavity, sepsis, multi-organ failure and even death. In problematic locations like the rectum, the leakage rate has not yet fallen below 10 %. Such a life-threatening condition is the result of impaired healing in the anastomotic wound. It is still vital to find innovative strategies and techniques in order to support regeneration of the anastomotic wound. This paper reviews the surgical techniques and biomaterials used, tested or published. Electrospun nanofibers are introduced as a novel and potential material in gastrointestinal surgery. Nanofibers possess several, unique, physical and chemical properties, that may effectively stimulate cell proliferation and collagen production; a key requirement for the healed intestinal wound.

Composite 3D printed scaffold with structured electrospun nanofibers promotes chondrocyte adhesion and infiltration.

Additive manufacturing, also called 3D printing, is an effective method for preparing scaffolds with defined structure and porosity. The disadvantage of the technique is the excessive smoothness of the printed fibers, which does not support cell adhesion. In the present study, a 3D printed scaffold was combined with electrospun classic or structured nanofibers to promote cell adhesion. Structured nanofibers were used to improve the infiltration of cells into the scaffold. Electrospun layers were connected to 3D printed fibers by gluing, thus enabling the fabrication of scaffolds with unlimited thickness. The composite 3D printed/nanofibrous scaffolds were seeded with primary chondrocytes and tested in vitro for cell adhesion, proliferation and differentiation. The experiment showed excellent cell infiltration, viability, and good cell proliferation. On the other hand, partial chondrocyte dedifferentiation was shown. Other materials supporting chondrogenic differentiation will be investigated in future studies.

The combination of nanofibrous and microfibrous materials for enhancement of cell infiltration and in vivo bone tissue formation.

Fibrous scaffolds are desired in tissue engineering applications for their ability to mimic extracellular matrix. In this study we compared fibrous scaffolds prepared from polycaprolactone using three different fabrication methods, electrospinning (ES), electro-blowing and melt-blown combined with ES. Scaffolds differed in morphology, fiber diameters and pore sizes. Mesenchymal stem cell adhesion, proliferation and osteogenic differentiation on scaffolds was evaluated. The most promising scaffold was shown to be melt-blown in combination with ES which combined properties of both technologies. Microfibers enabled good cell infiltration and nanofibers enhanced cell adhesion. This scaffold was used for further testing in critical sized defects in rabbits. New bone tissue formation occurred from the side of the treated defects, compared to a control group where only fat tissue was present. Polycaprolactone fibrous scaffold prepared using a combination of melt-blown and ES technology seems to be promising for bone regeneration. The practical application of results is connected with enormous production capacity and low cost of materials produced by melt-blown technology, compared to other bone scaffold fabrication methods.

Nanofibrous polycaprolactone scaffolds with adhered platelets stimulate proliferation of skin cells.

OBJECTIVES: Faulty wound healing is a global healthcare problem. Chronic wounds are generally characterized by a reduction in availability of growth factors. New strategies are being developed to deliver growth factors more effectively. METHODS: In this study, we introduced electrospun scaffolds composed of polycaprolactone (PCL) nanofibers functionalized with adhered platelets, as a source of numerous growth factors. Three concentrations of platelets were immobilized to nanofibrous scaffolds by simple adhesion, and their influence on adhesion, proliferation and metabolic activity of seeded cells (murine fibroblasts, keratinocytes and melanocytes) was investigated. RESULTS: The data obtained indicated that presence of platelets significantly promoted cell spreading, proliferation and metabolic activity in all the skin-associated cell types. There were no significant differences among tested concentrations of platelets, thus even the lowest concentration sufficiently promoted proliferation of the seeded cells. CONCLUSIONS: Such complex stimulation is needed for improved healing of chronic wounds. However, the nanofibrous system can be used not only as a skin cover, but also in broader applications in regenerative medicine.

Polycaprolactone foam functionalized with chitosan microparticles - a suitable scaffold for cartilage regeneration.

For biodegradable porous scaffolds to have a potential application in cartilage regeneration, they should enable cell growth and differentiation and should have adequate mechanical properties. In this study, our aim was to prepare biocompatible scaffolds with improved biomechanical properties. To this end, we have developed foam scaffolds from poly-epsilon-caprolactone (PCL) with incorporated chitosan microparticles. The scaffolds were prepared by a salt leaching technique from either 10 or 15 wt% PCL solutions containing 0, 10 and 20 wt% chitosan microparticles, where the same amount and size of NaCl was used as a porogen in all the cases. PCL scaffolds without and with low amounts of chitosan (0 and 10 wt% chitosan) showed higher DNA content than scaffolds with high amounts of chitosan during a 22-day experiment. 10 wt% PCL with 10 and 20 wt% chitosan showed significantly increased viscoelastic properties compared to 15 wt% PCL scaffolds with 0 and 10 wt% chitosan. Thus, 10 wt% PCL scaffolds with 0 wt% and 10 wt% chitosan are potential scaffolds for cartilage regeneration.

Collagen/hydroxyapatite scaffold enriched with polycaprolactone nanofibers, thrombocyte-rich solution and mesenchymal stem cells promotes regeneration in large bone defect in vivo.

A three-dimensional scaffold of type I collagen and hydroxyapatite enriched with polycaprolactone nanofibers (Coll/HA/PCL), autologous mesenchymal stem cells (MSCs) in osteogenic media, and thrombocyte-rich solution (TRS) was an optimal implant for bone regeneration in vivo in white rabbits. Nanofibers optimized the viscoelastic properties of the Coll/HA scaffold for bone regeneration. MSCs and TRS in the composite scaffold improved bone regeneration. Three types of Coll/HA/PCL scaffold were prepared: an MSC-enriched scaffold, a TRS-enriched scaffold, and a scaffold enriched with both MSCs and TRS. These scaffolds were implanted into femoral condyle defects 6 mm in diameter and 10-mm deep. Untreated defects were used as a control. Macroscopic and histological analyses of the regenerated tissue from all groups were performed 12 weeks after implantation. The highest volume and most uniform distribution of newly formed bone occurred in defects treated with scaffolds enriched with both MSCs and TRS compared with that in defects treated with scaffolds enriched by either component alone. The modulus of elasticity in compressive testing was significantly higher in the Coll/HA/PCL scaffold than those without nanofibers. The composite Coll scaffold functionalized with PCL nanofibers and enriched with MSCs and TRS appears to be a novel treatment for bone defects.

Elastic three-dimensional poly (ε-caprolactone) nanofibre scaffold enhances migration, proliferation and osteogenic differentiation of mesenchymal stem cells.

OBJECTIVES: We prepared 3D poly (ε-caprolactone) (PCL) nanofibre scaffolds and tested their use for seeding, proliferation, differentiation and migration of mesenchymal stem cell (MSCs). MATERIALS AND METHODS: 3D nanofibres were prepared using a special collector for common electrospinning; simultaneously, a 2D PCL nanofibre layer was prepared using a classic plain collector. Both scaffolds were seeded with MSCs and biologically tested. MSC adhesion, migration, proliferation and osteogenic differentiation were investigated. RESULTS: The 3D PCL scaffold was characterized by having better biomechanical properties, namely greater elasticity and resistance against stress and strain, thus this scaffold will be able to find broad applications in tissue engineering. Clearly, while nanofibre layers of the 2D scaffold prevented MSCs from migrating through the conformation, cells infiltrated freely through the 3D scaffold. MSC adhesion to the 3D nanofibre PCL layer was also statistically more common than to the 2D scaffold (P < 0.05), and proliferation and viability of MSCs 2 or 3 weeks post-seeding, were also greater on the 3D scaffold. In addition, the 3D PCL scaffold was also characterized by displaying enhanced MSC osteogenic differentiation. CONCLUSIONS: We draw the conclusion that all positive effects observed using the 3D PCL nanofibre scaffold are related to the larger fibre surface area available to the cells. Thus, the proposed 3D structure of the nanofibre layer will find a wide array of applications in tissue engineering and regenerative medicine.

Raster image correlation spectroscopy as a novel tool to study interactions of macromolecules with nanofiber scaffolds.

Dynamic processes such as diffusion and binding/unbinding of macromolecules (e.g. growth factors or nutrients) are crucial parameters for the design and application of effective artificial tissue materials. Here, dynamics of selected macromolecules were studied in two different composite tissue engineering scaffolds containing an electrospun nanofiber mesh (polycaprolactone or hydrophobically plasma modified polyvinylalcohol-chitosan) encapsulated in agarose hydrogels by a conventional approach fluorescence recovery after photobleaching (FRAP) and a novel technique, raster image correlation spectroscopy (RICS). The two approaches are compared, and it is shown that FRAP is unable to determine processes occurring at low molecular concentrations, especially accurately separating binding/unbinding from diffusion, and its results depend on the concentration of the studied molecules. RICS measures processes of single molecules and, because of its multiple adjustable timescales, can distinguish whether diffusion or binding controls molecular movement and separates fast diffusion from slow transient binding. In addition, RICS provides a robust read-out parameter quantifying binding affinity. Finally, the combination of FRAP and RICS helps to characterize diffusion and binding of macromolecules in tested artificial tissues better, and therefore predicts the behavior of biologically active molecules in these materials for medical applications.

Optimized conditions for mesenchymal stem cells to differentiate into osteoblasts on a collagen/hydroxyapatite matrix.

Collagen/hydroxyapatite (HA) composite scaffolds are known to be suitable scaffolds for seeding with mesenchymal stem cells (MSCs) differentiated into osteoblasts and for the in vitro production of artificial bones. However, the optimal collagen/HA ratio remains unclear. Our study confirmed that a higher collagen content increased scaffold stiffness but that a greater stiffness was not sufficient for bone tissue formation, a complex process evidently also dependent on scaffold porosity. We found that the scaffold pore diameter was dependent on the concentration of collagen and HA and that it could play a key role in cell seeding. In conclusion, the optimal scaffold for new bone formation and cell proliferation was found to be a composite scaffold formed from 50 wt % HA in 0.5 wt % collagen I solution.

Immobilization of thrombocytes on PCL nanofibres enhances chondrocyte proliferation in vitro.

OBJECTIVES: The aim of this study was to develop functionalized nanofibres as a simple delivery system for growth factors (GFs) and make nanofibre cell-seeded scaffold implants a one-step intervention. MATERIALS AND METHODS: We have functionalized polycaprolactone (PCL) nanofibres with thrombocytes adherent on them. Immobilized, these thrombocytes attached to nanofibre scaffolds were used as a nanoscale delivery system for native (autologous) proliferation and differentiation factors, in vitro. Pig chondrocytes were seeded on the thrombocyte-coated scaffolds and levels of proliferation and differentiation of these cells were compared with those seeded on non-coated scaffolds. RESULTS: Immobilized thrombocytes on PCL nanofibres effectively enhanced chondrocyte proliferation due to time-dependent degradation of thrombocytes and release of their GFs. CONCLUSIONS: These simply functionalized scaffolds present new possibilities for nanofibre applications, as smart cell scaffolds equipped with a GF delivery tool.

Non-woven PGA/PVA fibrous mesh as an appropriate scaffold for chondrocyte proliferation.

Non-woven textile mesh from polyglycolic acid (PGA) was found as a proper material for chondrocyte adhesion but worse for their proliferation. Neither hyaluronic acid nor chitosan nor polyvinyl alcohol (PVA) increased chondrocyte adhesion. However, chondrocyte proliferation suffered from acidic byproducts of PGA degradation. However, the addition of PVA and/or chitosan into a wet-laid non-woven textile mesh from PGA improved chondrocyte proliferation seeded in vitro on the PGA-based composite scaffold namely due to a diminished acidification of their microenvironment. This PVA/PGA composite mesh used in combination with a proper hydrogel minimized the negative effect of PGA degradation without dropping positive parameters of the PGA wet-laid non-woven textile mesh. In fact, presence of PVA and/or chitosan in the PGA-based wet-laid non-woven textile mesh even advanced the PGA-based wet-laid non-woven textile mesh for chondrocyte seeding and artificial cartilage production due to a positive effect of PVA in such a scaffold on chondrocyte proliferation.

Quality of newly formed cartilaginous tissue in defects of articular surface after transplantation of mesenchymal stem cells in a composite scaffold based on collagen I with chitosan micro- and nanofibres.

The aim of this study was to evaluate macroscopically, histologically and immunohistochemically the quality of newly formed tissue in iatrogenic defects of articular cartilage of the femur condyle in miniature pigs treated with the clinically used method of microfractures in comparison with the transplantation of a combination of a composite scaffold with allogeneic mesenchymal stem cells (MSCs) or the composite scaffold alone. The newly formed cartilaginous tissue filling the defects of articular cartilage after transplantation of the scaffold with MSCs (Group A) had in 60 % of cases a macroscopically smooth surface. In all lesions after the transplantation of the scaffold alone (Group B) or after the method of microfractures (Group C), erosions/fissures or osteophytes were found on the surface. The results of histological and immunohistochemical examination using the modified scoring system according to O'Driscoll were as follows: 14.7+/-3.82 points after transplantations of the scaffold with MSCs (Group A); 5.3+/-2.88 points after transplantations of the scaffold alone (Group B); and 5.2+/-0.64 points after treatment with microfractures (Group C). The O'Driscoll score in animals of Group A was significantly higher than in animals of Group B or Group C (p<0.0005 both). No significant difference was found in the O'Driscoll score between Groups B and C. The treatment of iatrogenic lesions of the articular cartilage surface on the condyles of femur in miniature pigs using transplantation of MSCs in the composite scaffold led to the filling of defects by a tissue of the appearance of hyaline cartilage. Lesions treated by implantation of the scaffold alone or by the method of microfractures were filled with fibrous cartilage with worse macroscopic, histological and immunohistochemical indicators.

The pi-helix formation between Asp369 and Thr375 as a key factor in E1-E2 conformational change of Na+/K+-ATPase.

Molecular modeling of the H4-H5-loop of the alpha2 isoform of Na+/K+-ATPase in the E1 and E2 conformations revealed that twisting of the nucleotide (N) domain toward the phosphorylation (P) domain is connected with the formation of a short pi-helix between Asp369 and Thr375. This conformational change close to the hinge region between the N-domain and the P-domain could be an important event leading to a bending of the N-domain by 64.7 degrees and to a shortening of the distance between the ATP binding site and the phosphorylation site (Asp369) by 1.22 nm from 3.22 nm to 2.00 nm. It is hypothesized that this shortening mechanism is involved in the Na+-dependent formation of the Asp369 phospho-intermediate as part of the overall Na+/K+-ATPase activity.

Use of allogenic stem cells for the prevention of bone bridge formation in miniature pigs.

This study appears from an experiment previously carried out in New Zealand white rabbits. Allogenic mesenchymal stem cells (MSCs) were transplanted into an iatrogenically-created defect in the lateral section of the distal physis of the left femur in 10 miniature pigs. The right femur with the same defect served as a control. To transfer MSCs, a freshly prepared porous scaffold was used, based on collagen and chitosan, constituting a compact tube into which MSCs were implanted. The pigs were euthanized four months after the transplantation. On average, the left femur with transplanted MSCs grew more in length (0.56+/-0.14 cm) compared with right femurs with physeal defect without transplanted MSCs (0.14+/-0.3 cm). The average angular (valgus) deformity of the left femur had an angle point of 0.78 degrees , following measurement and X-ray examination, whereas in the right femur without transplantation it was 3.7 degrees. The initial results indicate that preventive transplantation of MSCs into a physeal defect may prevent valgus deformity formation and probably also reduce disorders of the longitudinal bone growth. This part of our experiment is significant in the effort to advance MSCs application in human medicine by using pig as a model, which is the next step after experimenting on rabbits.