Implementing tissue engineering and regenerative medicine solutions in medical implants.

BACKGROUND: Surgical implants are widely used in the medical field but their long-term performance is limited due to failure of integration with tissues. This manuscript describes very well-known problems associated with implants and discusses novel solutions used in tissue engineering and regenerative medicine that can be implemented in this uncommonly discussed medical area. SOURCES OF DATA: General and medical literature describing modifications of medical and surgical implants, biofunctionalization, tissue engineering and regenerative medicine. AREAS OF AGREEMENT: Procedures for surgical implantation have grown substantially in the last few decades and provided improved quality of life for patients, regardless of area of implantation and device type and purpose. AREAS OF CONTROVERSY: In general, implants fail because of lack of long-term integration with the surrounding tissues. Implant manufacturers have not addressed implant failure from the point of view of biointegration. In addition, some medical practitioners are inclined to treat implant failure by using anti-infection methods to prevent bacterial adhesion. However, both approaches are conceptually limited, as discussed in this manuscript. GROWING POINTS: Implantation in the future will not be limited to medically needed procedures but also to a growing number of cosmetic body transformation procedures, which may include perceived 'improved implant functions' over natural tissues or organs. An additional trend is that implant procedures are being progressively performed in younger individuals. AREAS TIMELY FOR DEVELOPING RESEARCH: Current implants generally do not allow the physician to have controlled long-term access to internal tissues in contact with the implants, for example to release specific compounds when medically needed to the problem area.

Characterization of cultured epithelial cells using a novel technique not requiring enzymatic digestion for subculturing.

Our laboratory had developed a methodology to expand epithelial cells in culture by growing keratinocyte monolayers, under large volumes of medium that produces large numbers of keratinocytes that leave the monolayer and move into suspension. The cells have been defined as epithelial Pop Up Keratinocytes or ePUKs cells and appear to be highly suitable for clinical applications. In this publication we extend the characterization of the cells with a detailed analysis of the capabilities of the monolayer of a single culture flask to produce, over time, ePUK cells. The cells were characterized using standard epithelial markers for proliferation and differentiation. Analysis of morphology of the monolayer formed and total number of cells produced is presented for a variety of human epithelial cell strains. These keratinocytes provide an additional controlled human cell system for investigation of the mechanisms regulating epithelia cell growth and differentiation and since they are produced in large numbers, they are highly suitable for use in epithelial cell banking.

Characterization of a unique technique for culturing primary adult human epithelial progenitor/"stem cells".

BACKGROUND: Primary keratinocytes derived from epidermis, oral mucosa, and urothelium are used in construction of cell based wound healing devices and in regenerative medicine. This study presents in vitro technology that rapidly expands keratinocytes in culture by growing monolayers under large volumes of serum-free, essential fatty acid free, low calcium medium that is replaced every 24 hrs. METHODS: Primary cell cultures were produced from epidermal skin, oral mucosa and ureter by trypsinization of tissue. Cells were grown using Epilife medium with growth factors under high medium volumes. Once densely confluent, the keratinocyte monolayer produced cells in suspension in the overlying medium that can be harvested every 24 hrs. over a 7-10 day period. The cell suspension (approximately 8 X 105 cells/ml) is poured into a new flask to form another confluent monolayer over 2-4 days. This new culture, in turn produced additional cell suspensions that when serially passed expand the cell strain over 2-3 months, without the use of enzymes to split the cultures. The cell suspension, called epithelial Pop Up Keratinocytes (ePUKs) were analyzed for culture expansion, cell size and glucose utilization, attachment to carrier beads, micro-spheroid formation, induction of keratinocyte differentiation, and characterized by immunohistochemistry. RESULTS: The ePUKs expanded greatly in culture, attached to carrier beads, did not form micro-spheroids, used approximately 50% of medium glucose over 24 hrs., contained a greater portion of smaller diameter cells (8-10 microns), reverted to classical appearing cultures when returned to routine feeding schedules (48 hrs. and 15 ml/T-75 flask) and can be differentiated by either adding 1.2 mM medium calcium, or essential fatty acids. The ePUK cells are identified as cycling (Ki67 expressing) basal cells (p63, K14 expressing). CONCLUSIONS: Using this primary culture technique, large quantities of epithelial cells can be generated without the use of the enzyme trypsin to split the cultures. The cells are small in diameter and have basal cell progenitor/"stem" (P/SC) cell characteristics induced by daily feeding with larger than normal medium volumes. The ePUK epithelial cells have the potential to be used in regenerative medicine and for basic studies of epithelia P/SC phenotype.

Continuous delivery of biomaterials to the skin-percutaneous device interface using a fluid pump.

We have developed an in vitro culture system composed of organotypic human skin explants interfaced with titanium rods attached to a fluid pump. This device was designed to mimic the process of natural mucosa delivery at the point where a rigid, permanent object penetrates living skin. Full thickness human breast skin explants discarded from surgeries were cultured at different time points at the air-liquid interface. The skin specimens were punctured to fit at the bottom of hollow cylindrical titanium rods. Sodium lauryl sulfate (SLS) was delivered continuously to the specimens through the rods by using an attached fluid pump. Histological analysis of the skin explants as well as no-pump controls was then performed. Our results show substantial differences between controls, where no material was pumped at the interface of rod-skin, and specimens treated with SLS, indicating that the technique of pumping the material is effective in producing observable epithelial changes. These results suggest that an adaptation of this type of device may be useful for the treatment of complications arising from the contact between tissues and percutaneous devices in vivo.

Static adhesion of cancer cells to glass surfaces coated with glycosaminoglycans.

Using a previously described method for the functionalization of glass substrates with glycosaminoglycans (GAGs), in vitro experimental comparison of adhesion levels of cancer cells to glycosaminoglycan-modified substrates was performed with non-treated and heparin-treated human cancer cells of different metastatic activity. For both non-treated and heparin-treated cells, our results indicate that heparan sulfate is the preferred substrate for adhesion while keratan sulfate shows anti-adhesive properties. The observed net effect of heparin is a cell-dependent reduction in the adhesion figures. Overall, our results suggest that tissues with higher composition of heparan sulfate chains may be preferential metastatic targets and indicate that the effective use of heparin as anti-metastatic or anti-inflammatory agent may also depend on glycosaminoglycan composition of the affected organs.

Molecular dynamics studies show solvation structure of type III antifreeze protein is disrupted at low pH.

Antifreeze proteins are a class of biological molecules of interest in many research and industrial applications due to their highly specialized function, but there is little information of their stability and properties under varied pH derived from computational studies. To gain novel insights in this area, we conducted molecular dynamics (MD) simulations with the antifreeze protein 1KDF at varied temperatures and pH. Water solvation and H-bond formation around specific residues - ASN14, THR18 and GLN44 - involved in its antifreeze activity were extensively studied. We found that at pH1 there was a disruption in water solvation around the basal and the ice binding surfaces of the molecule. This was induced by a small change in the secondary structure propensities of some titrable residues, particularly GLU35. This change explains the experimentally observed reduction in antifreeze activity previously reported for this protein at pH1. We also found that THR18 showed extremely low H-bond formation, and that the three antifreeze residues all had very low average H-bond lifetimes. Our results confirm long-standing assumptions that these small, compact molecules can maintain their antifreeze activity in a wide range of pH, while demonstrating the mechanism that may reduce antifreeze activity at low pH. This aspect is useful when considering industrial and commercial use of antifreeze proteins subject to extreme pH environments, in particular in food industrial applications.

miRNAs in Tuberculosis: New Avenues for Diagnosis and Host-Directed Therapy.

Tuberculosis (TB) is one of the most fatal infectious diseases and a leading cause of mortality, with 95% of these deaths occurring in developing countries. The causative agent, Mycobacterium tuberculosis (Mtb), has a well-established ability to circumvent the host's immune system for its intracellular survival. microRNAs (miRNAs) are small, non-coding RNAs having an important function at the post-transcriptional level and are involved in shaping immunity by regulating the repertoire of genes expressed in immune cells. It has been established in recent studies that the innate immune response against TB is significantly regulated by miRNAs. Moreover, differential expression of miRNA in Mtb infection can reflect the disease progression and may help distinguish between active and latent TB infection (LTBI). These findings encouraged the application of miRNAs as potential biomarkers. Similarly, active participation of miRNAs in modulation of autophagy and apoptosis responses against Mtb opens an exciting avenue for the exploitation of miRNAs as host directed therapy (HDT) against TB. Nanoparticles mediated delivery of miRNAs to treat various diseases has been reported and this technology has a great potential to be used in TB. In reality, this exploitation of miRNAs as biomarkers and in HDT is still in its infancy stage, and more studies using animal models mimicking human TB are advocated to assess the role of miRNAs as biomarkers and therapeutic targets. In this review, we attempt to summarize the recent advancements in the role of miRNAs in TB as immune modulator, miRNAs' capability to distinguish between active and latent TB and, finally, usage of miRNAs as therapeutic targets against TB.

Protocol for serial cultivation of epithelial cells without enzymes or chemical compounds.

Deriving keratinocytes from epidermis or oral mucosa is a critical first step in the construction of cell-based tissue engineering and regenerative medicine applications. It would be advantageous to develop a methodology to grow adult somatic cells with maximum plasticity in a rapid fashion and in large numbers with minimal manipulation. With routine methods, keratinocytes are cultured in standard amount of medium and passaged with enzymes, and the confluence of the monolayer induces differentiation and eventual cell death. A protocol to expand keratinocytes in culture by growing keratinocyte in large numbers using a technique in which keratinocytes are released into the overlaying medium, effectively "popping-up" into suspension from the cell monolayer, is described in this chapter. This technique does not require the use of enzymes or chemical compounds for serial cultivation. The cells possess the ability of active cell proliferation at 100 % confluence over 1-2 months' time. Based on previous characterization reports, these are untransformed, normal keratinocytes that appear to be highly suitable for clinical applications.

Visible effects of rapamycin (sirolimus) on human skin explants in vitro.

In this manuscript, we report observations of the effects of rapamycin in an organotypic culture of human skin explants. The tissues were cultured for 5 days at the air-liquid interface or in submersed conditions with media with and without rapamycin at 2 nM concentration. Histological analysis of tissue sections indicated that rapamycin-treated samples maintained a better epidermal structure in the upper layers of the tissue than untreated samples, mostly evident when skin was cultured in submersed conditions. A significant decrease in the number of positive proliferative cells using the Ki67 antigen was observed when specimens were treated with rapamycin, in both air-liquid and submersed conditions but apoptosis differences between treated and untreated specimens, as seen by cleaved caspase-3 positive cells, were only observed in submersed specimens. Finally, a decrease and variability in the location in the expression of the differentiation marker involucrin and in E-cadherin were also evident in submersed samples. These results suggest that the development of topical applications containing rapamycin, instead of systemic delivery, may be a useful tool in the treatment of skin diseases that require reduction of proliferation and modulation or control of keratinocyte differentiation.

Tissue engineering of lips and muco-cutaneous junctions: in vitro development of tissue engineered constructs of oral mucosa and skin for lip reconstruction.

We report for the first time the fabrication of a three-dimensional tissue structure containing, in a continuous layer, the morphological features of a lip: epidermal skin, vermillion, and oral mucosa. This tissue engineered muco-cutaneous (M/C) equivalent was manufactured using human oral and skin keratinocytes grown on an acellular, nonimmunogenic dermal equivalent (AlloDerm(®)) to produce a tissue equivalent with similar anatomic and handling properties as native human lips. Confirmation of the structural composition of the construct was performed using routine histology and immunohistochemistry by identification of epithelial markers that are differentially expressed in separate anatomic areas of the lips. These full-thickness human lip skin equivalents can be used in surgical lip reconstruction in individuals suffering from lip loss from cancer, congenital deformations, and injuries after accidents. We propose this technique can be used as a general basis for tissue engineering of M/C junctions in other parts of the body, such as anus and vagina.

Physical characterization of mouse deep vein thrombosis derived microparticles by differential filtration with nanopore filters.

With the objective of making advancements in the area of pro-thrombotic microparticle characterization in cardiovascular biology, we present a novel method to separate blood circulating microparticles using a membrane-based, nanopore filtration system. In this qualitative study, electron microscopy observations of these pro-thrombotic mouse microparticles, as well as mouse platelets and leukocytes obtained using a mouse inferior vena cava ligation model of deep-vein thrombosis are presented. In particular, we present mouse microparticle morphology and microstructure using SEM and TEM indicating that they appear to be mostly spherical with diameters in the 100 to 350 nm range. The nanopore filtration technique presented is focused on the development of novel methodologies to isolate and characterize blood circulating microparticles that can be used in conjunction with other methodologies. We believe that determination of microparticle size and structure is a critical step for the development of reliable assays with clinical or research application in thrombosis and it will contribute to the field of nanomedicine in thrombosis.

Autologous Cell Delivery to the Skin-Implant Interface via the Lumen of Percutaneous Devices in vitro.

Induced tissue regeneration around percutaneous medical implants could be a useful method to prevent the failure of the medical device, especially when the epidermal seal around the implant is disrupted and the implant must be maintained over a long period of time. In this manuscript, a novel concept and technique is introduced in which autologous keratinocytes were delivered to the interfacial area of a skin-implant using the hollow interior of a fixator pin as a conduit. Full thickness human skin explants discarded from surgeries were cultured at the air-liquid interface and were punctured to fit at the bottom of hollow cylindrical stainless steel fixator pins. Autologous keratinocytes, previously extracted from the same piece of skin and cultured separately, were delivered to the specimens thorough the interior of the hollow pins. The delivered cells survived the process and resembled undifferentiated epithelium, with variations in size and shape. Viability was demonstrated by the lack of morphologic evidence of necrosis or apoptosis. Although the cells did not form organized epithelial structures, differentiation toward a keratinocyte phenotype was evident immunohistochemically. These results suggest that an adaptation of this technique could be useful for the treatment of complications arising from the contact between skin and percutaneous devices in vivo.

Bioengineering the skin-implant interface: the use of regenerative therapies in implanted devices.

This discussion and review article focuses on the possible use of regenerative techniques applied to the interfaces between skin and medical implants. As is widely known, the area of contact between an implant and the skin--the skin-implant interface--is prone to recurrent and persistent problems originated from the lack of integration between the material of the implant and the skin. Producing a long-term successful biointerface between skin and the implanted device is still an unsolved problem. These complications have prevented the development of advanced prosthetics and the evolution of biointegrated devices with new technologies. While previous techniques addressing these issues have relied mostly on the coating of the implants or the modification of the topology of the devices, recent in vitro developed techniques have shown that is possible to introduce biocompatible and possibly regenerative materials at the skin-device interface. These techniques have also shown that the process of delivering the materials has biological effects on the skin surrounding the implant, thus converting bioinert into bioactive, dynamic interfaces. Given that the best clinical outcome is the long-term stabilization and integration of the soft tissue around the implant, this article presents the basis for the selection of regenerative materials and therapies for long-term use at the skin-device interface, with focus on the use of natural biopolymers and skin cell transplantation.

Improved preservation of the tissue surrounding percutaneous devices by hyaluronic acid and dermatan sulfate in a human skin explant model.

Cellular apoptosis and proliferation was analyzed in an in vitro culture system of organotypic human skin explants in the presence or absence of external fixator pins. The effect on the tissues of a mixture of hyaluronic acid and dermatan sulfate (HA+DS) delivered at the skin-pin interface was also studied. After 2 weeks in culture, skin specimens interfaced with fixator pins showed increased keratinocyte apoptosis and proliferation compared to specimens without fixator pins. Simultaneously, a relative reduction of apoptosis and proliferation was observed in specimens treated with the HA+DS mixture, regardless of fixation pin presence. In addition, the HA+DS mixture appeared to help in the preservation of the epidermal basal membrane. It is concluded that in this in vitro model, fixator pins induce keratinocyte apoptosis and hyperproliferation, which are reduced in the presence of the HA+DS mixture. These methods may be useful for a better maintenance of the soft tissue surrounding percutaneous devices in vivo.

In vivo electrical conductivity across critical nerve gaps using poly(3,4-ethylenedioxythiophene)-coated neural interfaces.

BACKGROUND: Bionic limbs require sensitive, durable, and physiologically relevant bidirectional control interfaces. Modern central nervous system interfacing is high risk, low fidelity, and failure prone. Peripheral nervous system interfaces will mitigate this risk and increase fidelity by greatly simplifying signal interpretation and delivery. This study evaluates in vivo relevance of a hybrid peripheral nervous system interface consisting of biological acellular muscle scaffolds made electrically conductive using poly(3,4-ethylenedioxythiophene). METHODS: Peripheral nervous system interfaces were tested in vivo using the rat hind-limb conduction-gap model for motor (peroneal) and sensory (sural) nerves. Experimental groups included acellular muscle, iron(III) chloride-treated acellular muscle, and poly(3,4-ethylenedioxythiophene) polymerized on acellular muscle, each compared with intact nerve, autogenous nerve graft, and empty (nonreconstructed) nerve gap controls (n=5 for each). Interface lengths tested included 0, 5, 10, and 20 mm. Immediately following implantation, the interface underwent electrophysiologic characterization in vivo using nerve conduction studies, compound muscle action potentials, and antidromic sensory nerve action potentials. RESULTS: Both efferent and afferent electrophysiology demonstrates acellular muscle-poly(3,4-ethylenedioxythiophene) interfaces conduct physiologic action potentials across nerve conduction gaps of at least 20 mm with amplitude and latency not differing from intact nerve or nerve grafts, with the exception of increased velocity in the acellular muscle-poly(3,4-ethylenedioxythiophene) interfaces. CONCLUSIONS: Nonmetallic, biosynthetic acellular muscle-poly(3,4-ethylenedioxythiophene) peripheral nervous system interfaces both sense and stimulate physiologically relevant efferent and afferent action potentials in vivo. This demonstrates their relevance not only as a nerve-electronic coupling device capable of reaching the long-sought goal of closed-loop neural control of a prosthetic limb, but also in a multitude of other bioelectrical applications.

Novel organotypic cultures of human skin explants with an implant-tissue biomaterial interface.

A novel in vitro culture system of organotypic human skin explants interfacing with external fixator pins is presented. The system was used to observe changes in skin morphology on the skin at the pin interface. To evaluate the performance of this novel system, histological analysis of human skin explants cultured for 5 days at the air-liquid interface was performed. Compared to control explants, specimens interfaced with pins (treated or not with a physiological saline solution) showed a deteriorating basal layer, a disappearing stratum spinosum and increased lost of elastic fibers in the dermis. The model system makes it possible to perform rapid, repeatable studies of living skin response to chronically implanted materials and devices.

In vitro integration of human skin dermis with porous cationic hydrogels.

Porous poly(DMAA-co-AMTAC) hydrogels, fabricated using the inverted colloid crystal method, were used to observe their integration with human skin. Full thickness human breast skin explants discarded from surgeries were cultured for up to 10days at the air-liquid interface using a Transwell culture system. Cylindrical, disk- or other shaped hydrogels were placed inside the skin explants fitting punctures produced by punch biopsies or scalpels and full section histological analysis of the skin explants with the inserted hydrogel was then performed. In addition, separated hydrogels were cultured up to 7days with human fibroblasts. The results indicate that poly(DMAA-co-AMTAC) hydrogels induce substantial extracellular matrix material deposition, maintain dermal integrity in the contact areas with the skin and permit dermal fibers to integrate into the hydrogel pores. Different types of cells remaining in the explants migrated into the hydrogels pores, including red blood cells. Fibroblasts adhered to and colonized separately cultured hydrogels. We plan to use this type of soft material as an interface to permit skin integration with percutaneous devices in contact with skin.

Quantitation of surface-reducing-end covalently bound polysaccharides via hydrazinolysis and deamination.

Depending on the method of deposition, reactive sites of polysaccharides on substrates may not be available when their reducing ends have been used to covalently bind them to the substrates. Here we present a method that allows surface density measurements of reducing-end covalently bound polysaccharides in a procedure that cleaves the polysaccharide chain from the surface via hydrazinolysis and deamination, leaving on the surface a disaccharide that is later radiolabeled with an aldehyde in a reaction with enamine formation. The method described has the advantage that it may be used with any polysaccharide patterned to any surface exposing an amino-terminated monolayer by reductive amination of their galactosamine or glucosamine repeating units. We illustrate the technique with the quantitation of glycosaminoglycans (GAGs) on silanized glass surfaces.

In situ polymerization of a conductive polymer in acellular muscle tissue constructs.

We present a method to chemically deposit a conductive polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), on acellularized muscle tissue constructs. Morphology and structure of the deposition was characterized using optical and scanning electron microscopies (SEM). The micrographs showed elongated, smooth, tubular PEDOT structures completely penetrating and surrounding the tissue fibers. The chemical polymerization was performed using iron chloride, a mild oxidizer. Remaining iron and chlorine in the tissue constructs were reduced to acceptable metabolic levels, while preserving the structural integrity of the tissue. We expect that these acellular, polymerized tissue implants will remain essentially unmodified in cellular environments in vitro and in vivo because of the chemical and thermal stability of the PEDOT polymer depositions. Our results indicate that in situ polymerization occurs throughout the tissue, converting it into an extensive acellular, non-antigenic substrate of interest for in vivo experiments related to nerve repair and bioartificial prosthesis. We expect these conducting polymer scaffolds to be useful for direct integration with electronically and ionically active tissues.

Deposition of patterned glycosaminoglycans on silanized glass surfaces.

We report the robust attachment of glycosaminoglycans (GAGs) on silanized glass surfaces. Depositions were performed both by immersion and by application of a pattern by means of microcontact printing. Immunofluorescence assays were performed to verify the deposition and the quality of the patterns. In addition, AFM studies of the coated surfaces were performed in order to study some physical characteristics of the deposited GAGs layers. These results may prove useful for the characterization of the mechanical properties of GAGs in the glycocalyx and its relation with cellular migration.