Peter E. Butler

Artificial nerve conduits in peripheral-nerve repair

Injuries to the nervous system are the result of mechanical, thermal, chemical or congenital pathologies and, if function is not restored, they lead to loss of muscle function, pain and impaired sensation. Current treatment modalities essentially coapt the two nerves ends together or place a nerve graft between the cut ends. However, clinical results have never been optimal, and therefore a quest for better options has taken place. In this review article we look at the synthetic and biomimetic options currently being tested as potential nerve grafts.

Tissue engineering: revolution and challenge in auricular cartilage reconstruction.

Summary: External ear reconstruction for congenital deformity such as microtia or following trauma remains one of the greatest challenges for reconstructive plastic surgeons. The problems faced in reconstructing the intricate ear framework are highly complex. A durable, inert material that is resistant to scar contracture is required. To date, no material, autologous or prosthetic, is available that perfectly mimics the shapely elastic cartilage found in the ear. Current procedure involves autologous costal cartilage that is sculpted to create a framework for the overlying soft tissues. However, this is associated with donor-site morbidity, and few surgeons worldwide are skilled in the techniques required to obtain excellent results. Various alloplastic materials have therefore been used as a framework. However, a degree of immunogenicity and infection and extrusion are inevitable, and results are often disappointing. Tissue-engineered cartilage is an alternative approach but, despite significant progress in this area, many problems remain. These need to be addressed before routine clinical application will become possible. The current tissue-engineered options are fragile and inflexible. The next generation of auricular cartilage engineering is promising, with smart materials to enhance cell growth and integration, and the application of stem cells in a clinical setting. More recently, the authors team designed the worlds first entirely synthetic trachea composed of a novel nanocomposite material seeded with the patients own stem cells. This was successfully transplanted in a patient at the Karolinska Hospital in Sweden and may translate into a tissue-engineered auricle in the future.

Induction of Tolerance to an Allogeneic Skin Flap Transplant in a Preclinical Large Animal Model

Clinical composite tissue allotransplantation can adequately reconstruct defects that are not possible by other means. However, immunosuppressant toxicity limits the use of these techniques. Clinical attempts to reduce the amount of immunosuppression required by induction of an immunologically permissive state have so far been unsuccessful. The aim of this study was to induce tolerance in a preclinical large animal model. Donor hematopoietic stem cell (HSC) engraftment was induced by T-cell depletion, irradiation, and a short course of cyclosporine administered to the recipient, along with a hematopoietic cell infusion from a single haplotype major histocompatibility complex (MHC) mismatched donor. Skin was then allotransplanted from the donor. Both primarily vascularized skin flaps and secondarily vascularized conventional skin grafts were allotransplanted to investigate if the mode of transplantation affected outcome. Control animals received the skin allotransplants without conditioning. Tolerance was defined as no evidence of rejection at 90 days following transplantation. Conventional skin grafts only achieved prolonged survival (<65 days) in HSC-engrafted animals (P < .01). In contrast, there was indefinite skin flap survival with the achievement of tolerance in HSC-engrafted animals; this was confirmed on histology with donor-specific unresponsiveness on MLR and CML. Furthermore, a conventional skin donor graft subsequently applied to an animal tolerant to a skin flap was not rejected and did not trigger skin flap rejection. To our knowledge, this is the first time skin tolerance has been achieved across a MHC barrier in a large animal model. This is a significant step toward the goal of clinical skin tolerance induction.

Optimization of chondrocyte isolation and characterization for large-scale cartilage tissue engineering.

BACKGROUNDnAdvancements in cartilage tissue engineering have the potential to ameliorate facial and joint reconstructive surgery as we know it. The translation of inxa0vitro models of cartilage regeneration into clinical scenarios is the next phase of cartilage tissue engineering research. To engineer larger, more robust, and clinical relevant constructs, a great number of viable chondrocytic cells are needed. However, there is a paucity of literature concerning the most favorable method of chondrocyte isolation. Isolation methods are inconsistent, resulting in small yields and poor cell quality, and thus unreliable neocartilage formation. This study aimed to optimize the chondrocyte isolation protocol to give a maximum yield with optimal cell viability for the engineering of large cartilaginous constructs such as the human nose and ear.nnnMETHODSnWe employed several enzymes (pronase, dispase, hyaluronidase, and collagenase), enzyme concentrations, and digest lengths to digest freshly harvested ovine nasoseptal cartilage. We used automated trypan blue live/dead staining, immunofluorescent labeling of CD44, collagenase II, collagenase I, and Aggrecan, and alamarBlue to assess cell yield and viability.nnnRESULTSnIncubation length in enzymatic solutions had the greatest effect on cell viability, whereas concentrations of enzymes had a lesser effect. Isolated cells maintained their expression of chondrocyte-specific cell surface markers.nnnCONCLUSIONSnThe optimum incubation period was 10 h using collagenase at a 0.2% (w/v) solution. An average of 1-1.5xa0×xa010(6) cells could be harvested per gram of cartilage using this method.

Skin tolerance: in search of the Holy Grail

In 1943, Gibson and Medawar opened the modern era of transplantation research with a paper on the problem of skin allograft rejection. Ten years later Billingham, Brent and Medawar demonstrated that it was possible to induce selective immune acceptance of skin grafts in mice, a state of tolerance. After over six decades, however, the precise mechanism of skin allograft rejection remains still ill‐defined. Furthermore, it has not been possible to achieve reliably clinical tolerance allowing the widespread application of skin allotransplantation techniques. The first successful applications of skin allotransplantation have included the hand and face. However, complications from the chronic immunosuppression regimens limit the application of these techniques. Induction of tolerance to skin (and the other tissues in the allograft) would be the most effective way to overcome all these difficulties, but this is yet to be achieved reliably, stimulating some to look for other ways to surmount the current limitations. This paper summarizes alternatives to enlarge the scope of skin allotransplantation techniques, current understanding of mechanisms of skin rejection, and the utility and limitations of animal models used to study skin rejection and tolerance induction. Finally, manipulation strategies to achieve skin tolerance are outlined.

Systematic review of patient factors affecting adipose stem cell viability and function: implications for regenerative therapy

BackgroundThe applications for fat grafting have increased recently, within both regenerative and reconstructive surgery. Although fat harvesting, processing and injection techniques have been extensively studied and standardised, this has not had a big impact on the variability of outcome following fat grafting. This suggests a possible larger role of patient characteristics on adipocyte and adipose-derived stem cell (ADSC) viability and function. This systematic review aims to collate current evidence on the effect of patient factors on adipocyte and ADSC behaviour.MethodsA systematic literature review was performed using MEDLINE, Cochrane Library and EMBASE. It includes outcomes observed in in vitro analyses, in vivo animal studies and clinical studies. Data from basic science work have been included in the discussion to enhance our understanding of the mechanism behind ADSC behaviour.ResultsA total of 41 papers were included in this review. Accumulating evidence indicates decreased proliferation and differentiation potential of ADSCs with increasing age, body mass index, diabetes mellitus and exposure to radiotherapy and Tamoxifen, although this was not uniformly seen across all studies. Gender, donor site preference, HIV status and chemotherapy did not show a significant influence on fat retention. Circulating oestrogen levels have been shown to support both adipocyte function and graft viability. Evidence so far suggests no significant impact of total cholesterol, hypertension, renal disease, physical exercise and peripheral vascular disease on ADSC yield.ConclusionsA more uniform comparison of all factors highlighted in this review, with the application of a combination of tests for each outcome measure, is essential to fully understand factors that affect adipocyte and ADSC viability, as well as functionality. As these patient factors interact, future studies looking at adipocyte viability need to take them into consideration for conclusions to be meaningful. This would provide crucial information for surgeons when deciding appropriate volumes of lipoaspirate to inject, improve patient selection, and counsel patient expectations with regards to outcomes and likelihood for repeat procedures. An improved understanding will also assist in identification of patient groups that would benefit from graft enrichment and cryopreservation techniques.

The Use of Adipose Stem Cells in Cranial Facial Surgery

Craniofacial malformations, have devastating psychosocial implications for many adults and children and causes huge socioeconomic burden. Currently craniofacial defects require soft tissue transfer, bone grafting techniques or difficult procedures such as microvascular free flaps. Such tissues are often limited in quantity, their harvest causes secondary large donor site defects and they lack the capability to fully restore previous form and function. Stem cell technology is being utilised for various tissue and organs of the body and consequently surgeons are eager to transfer these principles for craniofacial surgery. Adipose derived stem cells (ADSCs) are an exciting stem cell source for craniofacial surgeons due to their easy and painless isolation, relatively large abundance and familiarity with the harvesting procedure. ADSCs also have multiple desirable properties including adipogenic, osteogenic and chondrogenic potential, enhancement of angiogenesis and immunodulatory function. Due to these advantageous characteristics, ASDCs have been explored to repair craniofacial bone, soft tissue and cartilage. The desirable characteristics of ADSCs for craniofacial surgical applications will be explained. We report the experimental and clinical studies that have explored the use of ADSCs for bone, cartilage and soft tissue craniofacial defects. We conclude by establishing the key questions that are preventing the clinical application of ADSCs for craniofacial surgery.

Biomechanical Characterisation of the Human Auricular Cartilages; Implications for Tissue Engineering

Currently, autologous cartilage provides the gold standard for auricular reconstruction. However, synthetic biomaterials offer a number of advantages for ear reconstruction including decreased donor site morbidity and earlier surgery. Critical to implant success is the material’s mechanical properties as this affects biocompatibility and extrusion. The aim of this study was to determine the biomechanical properties of human auricular cartilage. Auricular cartilage from fifteen cadavers was indented with displacement of 1xa0mm/s and load of 300xa0g to obtain a Young’s modulus in compression. Histological analysis of the auricle was conducted according to glycoprotein, collagen, and elastin content. The compression modulus was calculated for each part of the auricle with the tragus at 1.67xa0±xa00.61xa0MPa, antitragus 1.79xa0±xa00.56xa0MPa, concha 2.08xa0±xa00.70xa0MPa, antihelix 1.71xa0±xa00.63xa0MPa, and helix 1.41xa0±xa00.67xa0MPa. The concha showed to have a significantly greater Young’s Elastic Modulus than the helix in compression (pxa0<xa00.05). The histological analysis demonstrated that the auricle has a homogenous structure in terms of chondrocyte morphology, extracellular matrix and elastin content. This study provides new information on the compressive mechanical properties and histological analysis of the human auricular cartilage, allowing surgeons to have a better understanding of suitable replacements. This study has provided a reference, by which cartilage replacements should be developed for auricular reconstruction.

A polyhedral oligomeric silsesquioxane-based bilayered dermal scaffold seeded with adipose tissue-derived stem cells: in vitro assessment of biomechanical properties

BACKGROUNDnAlthough commercial skin substitutes are widely available, its use remains challenging at surgery and postoperatively. The high cost is also prohibitive. We designed and characterized a scaffold for dermal replacement, using advanced nanocomposite materials, which are known to have unique nanoscale features that enhance cellular behavior.nnnMETHODSnA bilayered scaffold was developed using the nanocomposite, polyhedral oligomeric silsesquioxane, incorporated into poly(caprolactone-urea)urethane, resulting in a mechanically robust bioabsorbable polymer; forming the inner layer, which was designed with a range of porosities. The removable outer layer contained nanosilver. Tensile testing, surface tension, permeability, and scanning electron microscopy were performed. Optimal pore morphology for cellular proliferation was elucidated through adipose tissue-derived stem cell culture and a cell viability assay. All tests were repeated on Integra Dermal Regeneration Template.nnnRESULTSnThe physical construct was easy to handle and clinically applicable. Macroporosity and permeability of scaffolds was demonstrated, confirmed by scanning electron microscopy. Both tensile strength and surface tension were comparable with skin; outer layer demonstrated hydrophobicity and inner layer showed hydrophilicity. Cell assay confirmed cellular proliferation onto the scaffold, comparable with Integra.nnnCONCLUSIONSnWe demonstrate that a porous bilayered dermal scaffold could form the basis of a new generation of skin substitute that is both mechanically robust and harbors the ability for enhancing cell regeneration.

Development of mechano-responsive polymeric scaffolds using functionalized silica nano-fillers for the control of cellular functions

We demonstrate an efficient method to produce mechano-responsive polymeric scaffolds which can alter cellular functions using two different functionalized (OH and NH2) silica nano-fillers. Fumed silica-hydroxyl and fumed silica-amine nano-fillers were mixed with a biocompatible polymer (POSS-PCU) at various wt% to produce scaffolds. XPS and mechanical testing demonstrate that bulk mechanical properties are modified without changing the scaffolds surface chemistry. Mechanical testing showed significant change in bulk properties of POSS-PCU scaffolds with an addition of silica nanofillers as low as 1% (P < 0.01). Scaffolds modified with NH2 silica showed significantly higher bulk mechanical properties compared to the one modified with the OH group. Enhanced cell adhesion, proliferation and collagen production over 14 days were observed on scaffolds with higher bulk mechanical properties (NH2) compared to those with lower ones (unmodified and OH modified) (P < 0.05) during in vitro analysis. This study provides an effective method of manufacturing mechano-responsive polymeric scaffolds, which can help to customize cellular responses for biomaterial applications.