Antonio Peramo

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.

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.

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.

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

BackgroundPrimary 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.MethodsPrimary 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 e pithelial P op U p K eratinocytes (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.ResultsThe 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).ConclusionsUsing 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.

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.

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.

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.

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.