Epiflex(®) a new decellularised human skin tissue transplant: manufacture and properties.

The manufacture and initial testing of a new human tissue transplant is described. Epiflex(®) is a human acellular dermis transplant that is manufactured from skin recovered from screened consenting donors according to validated and approved methods. The transplant is approved as a drug in Germany. The safety, stability and usability of the transplant are discussed with respect to the results of sterility, residual moisture content and rehydration tests. Histological and confocal laser scanning microscopy experiments and analysis of oxygen and water vapour permeability demonstrate that the native extracellular matrix structure and transport properties of human connective tissue are retained in the transplant. Results from initial clinical investigations suggest that Epiflex(®) can be used successfully in the treatment of burns, hypertrophic scars and as a transplant seeded with autologous dermal fibroblasts for soft-tissue regeneration in settings with wound healing problems following multi-modal treatments for sarcomas of the extremities.

[Influence of European regulations on quality, safety and availability of cell and tissue allografts in Germany]. / Einfluss europäischer Regulativa auf Qualität, Sicherheit und Verfügbarkeit allogener Zell- und Gewebetransplantate in Deutschland.

The transplantation of allogenic tissue (bone, cartilage, tendon, skin, amnion and special preparations such as demineralised bone matrix and acellular dermis) is an important component of the treatment of bone and soft tissue defects, particularly in traumatology and orthopaedic, reconstructive and plastic surgery. In Germany, the requirement for such tissue transplants is met by supply from local tissue banks (in particular bone banks) and a small number of regional and national tissue banks. These banks operate on the basis of the "Guidelines for Bone Banks" laid down by the German Chamber of Physicians, and of the German Drug Law (AMG). The 2004/23/EG guidelines issued by the European Parliament and ratified on 31/3/2004 define the quality and safety standards for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. These guidelines will have a major impact on all aspects of tissue banking and transplantation. In particular, the new guidelines will remove the possibility for local tissue banks to operate outside of national drug laws ( section sign 4 a [4]). The currently in draft law on "Quality and Safety of Human Tissues and Cells" ("Tissue Law") of the Federal Health Ministry seems to be heading in this direction, but it also includes possibilities for the continuation of local banks. An additional European guideline draft "Proposal for the regulation of advanced therapeutic medical products" is currently under discussion. This paper assesses the impact of these new pieces of legislation on the quality, safety and availability of human cell and tissue transplants in terms of the current situation and future prospects in Germany.

Sufficient penetration of peracetic acid into drilled human femoral heads.

Chemical sterilisation methods for musculoskeletal transplants have the problem of penetration into all tissue strata. The present study examined if a peracetic acid/ethanol solution penetrated to a sufficient extent into specifically prepared femoral heads. To this effect, 10 femoral heads have been provided with drillings (diameter 2 mm, depth 10 mm) at a distance of 15 mm (series B) and placed in a diffusion chamber with sterilisation solution. From an additional central drilling at the femoral neck junction, the sample drawing was made after 30 min each over a period of 4 h for the iodometric determination of peracetic acid (PAA) concentration. Ten femoral heads, which did contain only the central drilling, served as controls (series A). In 9 of the examined femoral heads of series A the defined minimum concentration of PAA of 0.2% (inactivation of bacteria, spores, fungi) has been clearly exceeded over the complete period of measurement. About 0.8% PAA (inactivation of viruses) was achieved within 4 h only with six femoral heads. Nine out of the 10 examined femoral heads in series B show a clearly improved penetration behaviour which was expressed in smaller standard deviations, a faster increase in concentration, as well as in higher starting and final concentrations (approx. 0.9%) of PAA. Previous drying in air leads to a faster penetration into the centre of the bone. Standardised drilling of de-cartilaged femoral heads creates favourable conditions for the penetration of the PAA sterilisation solution into the whole tissue and guarantees a sufficient inactivation of microorganisms.

Biomechanical comparison of human bone-patellar tendon-bone grafts after sterilization with peracetic acid ethanol.

Recent reports of disease transmission following ACL reconstruction with fresh-frozen non-sterilized allografts have highlighted the need for new sterilization techniques that do not impair the mechanical properties as it was shown for most of the current sterilization techniques. In this in-vitro biomechanical study, it was investigated if peracetic acid ethanol sterilization (PES) has any adverse effects on the mechanical properties of human bone-patellar tendon-bone grafts (BPTB). Paired human BPTB grafts either underwent PES or were used as fresh-frozen non-sterilized grafts. Viscoelastic properties (strain, creep) were analyzed during cyclic submaximal loading and mechanical properties were investigated during load-to-failure (LTF) testing. It was found that there were no differences in viscoelastic and mechanical properties between both groups. The findings of this study provide baseline data for future in vitro and in vivo analyses of this promising new sterilization technique for soft-tissue allografts.

Preservation of vein allograft viability during long-term storage.

BACKGROUND: The aim of this study was to compare the vein allograft viability following cryopreservation with that remaining after prolonged refrigerated storage. MATERIALS AND METHODS: Great saphenous vein biopsies had been cryopreserved, and the samples were divided into two matched groups and stored in tissue culture medium for 42 days at +4 degrees C, either with or without regular medium replacement. Each vein allograft was biopsied and assayed for viability every third day by the methyltetrazolium reduction assay. Viability indexes of vein allografts harvested from brain-dead multi-organ donors and from cadavers whose warm ischemic periods were maximally 24 h were also compared. RESULTS: Vein allografts stored for 42 days at +4 degrees C showed a similar viability (58.9 +/- 1.2%) to that of cryopreserved veins (59.7 +/- 2.3%). This was true even when cryopreserved and thawed allografts were subjected to 3 days of post-thaw incubation under presumably favorable conditions (58.7 +/- 1.6%). There was no viability index difference between the samples with medium replaced and not replaced and the specimens harvested from two different donor groups. CONCLUSIONS: Long-term storage of vein allografts at +4 degrees C is a valuable option for regular banking practice. Sufficient amounts can be procured from cadavers similar to tissue donors.

Quantitative reduction of methyl tetrazolium by fresh vein homograft biopsies in vitro is an index of viability.

OBJECTIVE: The color density of the methyl tetrazolium (MTT) test is proportional to mitochondrial enzyme activity thus reflecting cellular viability. The aim was to evaluate the MTT test as a viability assay for vein homograft studies. MATERIALS AND METHODS: Fresh intact vein samples were harvested during multi-organ procurement. The reliability of the MTT assay was tested by a fluorescent dye combination (1 microg/ml propidium iodide PI and 4 microM/ml SYTO-16 stains). The enzyme kinetics of the reaction was also investigated. The optimal reagent concentration, biopsy size and incubation period were established. RESULTS: There was a linear relationship between the vein homograft's weight and the pigment production activity. A nonspecific reaction (8.6%) was observed in negative controls. The MTT cleavage up to 0.1% (w/v) follows the Michaelis kinetics. The Michaelis constant (2,805 +/- 130 microM), the maximal velocity (196 +/- 2.2 x 10(-5 )microM s(-1)) and the velocity constant (6.98 +/- 0.2 x 10(-7) s(-1)) was calculated. The viability assessed by fluorescent dyes simultaneously visualized the live/dead cell ratio, which can be calculated by image analysis software. CONCLUSION: The use of MTT in colorimetric assays offers high sensitivity. The assay is simple, inexpensive, and reproducible in vein homograft studies.

Validation of the sterilization procedure of allogeneic avital bone transplants using peracetic acid-ethanol.

Different procedures are available to inactivate bacteria and fungi, including their spores, as well as viruses in human bone transplants. The most efficient methods are considered to be gamma irradiation and thermal inactivation as well as chemical sterilization methods like the peracetic acid-ethanol treatment (PES). Following national and international standards or draft standards, the antimicrobial effectiveness of this procedure was evaluated. Due to the standardizable size as well as the clinical relevance, defatted human spongiosa cuboids (15x15x15 mm) served as model system. After treatment with PES for 2 and 4 hours, respectively, the titre of living micro-organisms was determined in the supernatant and the cuboid. A reduction in the titre of viable micro-organisms below the detection level (reduction factor >5 log10) was already achieved after an incubation time of 2 hours (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Bacillus subtilis, Clostridium sporogenes, Mycobacterium terrae, Candida albicans as well as spores of Bacillus subtilis). No viable micro-organisms could be detected in any of the PES-treated test cuboids. Spores of Aspergillus niger were also completely inactivated. The PES procedure proved to be a reliable method for the sterilization of human bone transplants derived from spongiosa.

Comparison of the efficacy of virus inactivation methods in allogeneic avital bone tissue transplants.

Several procedures for inactivating viruses are used presently in the context of bone tissue transplants. Common methods used are gamma irradiation (25 kGy), treatment with moist heat (82.5 degrees C/15 min., lobator-sd2-system) as well as chemical sterilisation using peracetic acid-ethanol treatment (PES, 2% peracetic acid, 96% ethanol, Aqua [2:1:1], 200 mbar, agitation, 4 hours). Based on national and international guidelines, we tested the antivirucidal effectiveness of these methods in human bone transplants. Three enveloped viruses: human immunodeficiency virus type 2 (HIV-2), pseudorabies virus (PRV), bovine virus diarrhoea virus (BVDV), and three non-enveloped viruses were used: hepatitis A virus (HAV), poliovirus (PV-1), porcine/bovine parvovirus (PPV, BPV). Defatted spongiosa cuboids served as model in chemical treatment experiments, while cortical diaphyses were used in gamma irradiation experiments, and the effects of thermal treatment were tested in prepared femoral heads. The log(10) reduction was measured by cytopathogenic effects after virus titration (TCID(50)/mL). A dose of at least 33.9 kGy (bone model) at -30 +/- 5 degrees C was necessary to achieve a sufficient reduction (4 log(10) steps) of BPV, the most resistant one of all viruses investigated. Thermal treatment as well as PES treatment led to a reduction of virus titres by more than 4 log(10). Only HAV showed a reduction below 4 log(10) (2.87) with PES. After validation of the defatting step included for HAV-infected cells, a HAV-reduction of over 7 log(10) was found. All three sterilisation methods tested are recommended for bone transplant sterilisation, but only provided that additional safety measures (anamnestic informations, infectious serology, PCR in case of multiorgan donors) are taken.

Demineralized Bone Matrix-stimulated Bone Regeneration in Rats Enhanced by an Angiogenic Dipeptide Derivate.

A lysyl-proline derivate (LP) known to stimulate angiogenesis and formation of granulation tissue was tested as a local additive to allogeneic demineralized bone matrix (DBM) using a rat craniotomy model. Peracetic-acid sterilized DBM (10 mg/defect) was implanted into three groups of 45 animals each with 0, 6 and 20 microg LP. Subsequent evaluation was done by descriptive histology, histomorphometry, and determination of the calcium content of the explants 7, 14, 28, 42 and 84 days post-implantation. Grafting with DBM alone resulted in defect bridging by newly formed bone with incorporated DBM residues on day 84. Addition of LP to the implants caused an enhanced capillarization on day 14 and 28 as well as an enhanced mineralization on day 14, 28, 42 and 84. Both effects were dose-dependent. These data suggest that the local application of a synthetic angiogenic factor significantly improve bone regeneration in DBM-grafted trephine defects in rats. Thereby, they reinforce the opinion that early angiogenesis is crucial for a number of subsequent events in the bone regeneration process.

Quality issues in tissue banking: quality management systems - a review.

The situation in tissue banking changed radically and fundamentally at the beginning of the 1990s. The essential causes are on the one hand, the continually increasing demand for human cells and tissue and other biological material for clinical use and research, and on the other hand, the rapid progress in the medical, technical and natural sciences. Biotechnology in particular, has profited from this. Modern tissue banks could no longer be imagined without its methods.A consequence of these developments and a prerequisite for the fulfilment of the derived requirements is the necessity for national and international cooperation as well as the harmonisation of ethical principles and quality assurance standards and regulations (von Versen (1999) Ann Chir Gynaecol 88: 215-220). The introduction of an all-encompassing Quality Management System (QMS) is a suitable instrument for this purpose.After the presentation of explanations and definitions of quality terminology, this article describes the use of the international standard ISO 9000 as a general QMS, which embraces both the specific methodology as well as the general aspects of Quality Management (research and development, design control, education and training, documentation, traceability, management control, corrective action, etc.) in tissue banking. The individual elements of this system are explained and selected examples are described. The authors look upon this QMS as an indispensable instrument for harmonisation and international cooperation in tissue banking.Finally, the use of such a standard would be a positive sign to the regulatory authorities and the public that tissue banking is making a visible effort to introduce a world-class QMS in its operations.

Musculoskeletal tissue banking in Europe--regulations and quality assurance.

This paper is confined to the use of human musculoskeletal tissue in the treatment of patients. Its focus is on the safety and quality dimension of human tissue transplantation, including the ethical and legal aspects, the regulations and standards from the European perspective, quality assurance and quality management in tissue banking and as a special subject, tissue sterilisation and the validation of sterilisation methods.

Virus safety of avital bone tissue transplants: evaluation of sterilization steps of spongiosa cuboids using a peracetic acid-methanol mixture.

The aim of this study was to validate the virus-inactivating/eliminating capacity of the manufacturing process of spongiosa cuboids. Both the sterilization step with peracetic acid (PAA)/ethanol and the defatting step of bones with chloroform/methanol (2:1, v/v) were investigated. Relevant enveloped, non-enveloped, and model viruses belonging to different virus families were included in the investigation: human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), poliovirus (PV-1), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine virus diarrhoea virus (BVDV). Treatment of virus-spiked spongiosa cuboids for 4 hours at room temperature (RT) with 1% PAA/24% ethanol (PES) efficiently inactivated most viruses. Titres were reduced by more than 4 log(10)with the exception of HAV. The defatting step with chloroform/methanol reduced HAV titres by a factor of >/=7.0 log(10). From these results it can be concluded that the treatment of spongiosa cuboids with (i) chloroform/methanol and (ii) 1% PAA/24% ethanol solution leads to a virus-safe medicinal product.

Human immunodeficiency virus (HIV) inactivation of banked bone by gamma irradiation.

OBJECTIVE: The increasing number of patients with acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus(HIV)-positive carriers, poses difficulties when musculo-skeletal tissues are considered for banking in readiness for future clinical application. This study was conducted to test the actual yield of gamma irradiation on HIV infectivity, within HIV-infected bones. METHODS: The effect of gamma irradiation on bones containing T-cells chronically infected with HIV type I (HIV-I) was studied, in respect to inactivation of the virus. RESULTS: After exposure of the cell-free virus or infected T-cells to 2.5 megarads of gamma irradiation, the authors were able to demonstrate complete inactivation of the virus. CONCLUSIONS: It would appear from this study that gamma irradiation at this dose is sufficient to achieve clinical sterilisation of bones and facilitate their use for reconstructive procedures by eliminating the risk of HIV transmission to the recipient. Furthermore, when preparing bones for banking, this would also seem to be the method of choice in preventing the transmission of various strains of bacteria, fungi and other viruses.

Electron microscopic study on the effects of different preservation methods for meniscal cartilage.

OBJECTIVES: The study was conducted to evaluate different methods for preservation of meniscal cartilage. During the last decade, the use of allografts containing cartilage in various forms, such as: small osteochondral grafts, osteoarticular grafts, or whole joints, has gained popularity. Meniscal allografts in particular, can be used in cases of post-menisectomy syndrome, or in uni-compartmental osteoarthritis of the knee. The preservation methods used for the cartilaginous components of allografts would appear to influence the success rates, which vary between 30% and 85% according to different reports. Some of the methods of preservation currently in use include: deep-freezing in liquid nitrogen, freezing to -80 degrees C, gamma irradiation, and immersion in ethylene oxide. METHODS: In an attempt to find the best technique for cartilage preservation (in terms of preserving the ultra-structural properties prior to implantation), bovine meniscal cartilage was examined after preservation by: a) freezing to-80 degrees C; b) exposure to 2.5 Mrad gamma irradiation; or c) a combination of both. RESULTS: Electron microscopic studies were performed, with fresh menisci serving as controls. Our tests demonstrated that in all techniques, severe cellular damage was caused, whereas the collagen network retained its properties. CONCLUSION: The optimal method for the preservation of cartilage remains to be established.

[Tissue preservation in the DRG]. / Gewebekonservierung in der DDR.

The conservation of tissue in GDR is an example system of superregional supply with tissue transplants, which was established with the foundation of the Central Tissue Bank at the Charité in 1956 e. g. with the following arrangement of tissue banks in Leipzig and Rostock in 1966 and 1969. The concept of this tissue banks is characterized by means of decentralised production of tissue material under conditions of section and the central preparation, conservation and storage (chemical or radiation sterilization, conservation on base of freeze-drying (or freeze-storage), aseptic packing and storage by room temperature). This system allows the consistent disposal of 30 various nonvital tissue preparations. Until 1989 more than 170,000 allogeneic and xenogeneic tissue transplants have been produced in the tissue banks of country of which nearly 130,000 were used in 300 clinics.

[Clinical study on the application of demineralized bone matrix (DBM) in surgical orthodontics]. / Klinische Studie zum Einsatz von demineralisierter Knochenmatrix (DBM) in der Chirurgischen Stomatologie.

Demineralized bone matrix has been tested in a clinical study concerning the filling of bone defects in the surgical stomatology. It is reported on the clinical results and the X-ray evaluation of the replacement of bones by demineralized bone matrix. Fifty two implantations in the maxilla and mandible of 51 patients were carried out to be able to evaluate the efficacy of demineralized bone matrix.

[Methods for the preservation of soft tissue preparations for clinical use]. / Verfahren zur Herstellung von Weichteilpräparaten für die klinische Anwendung.

The demand for consistent supply of nonvital allogenous implants in tissue banks has the supposition for graft preservation. Besides of many logistic advantages the preservation methods are accompanied by the influence of original attributes, especially of biomechanic parameters in soft tissue preparations. A procedure of dura mater preparation, of fascia lata, tendons and skin is demonstrated which is preserving the required qualities of plasticity, softness and resistance. Biomechanic comparative investigations of other preservation procedures are done and first clinical results are demonstrated.

[Circular intrathoracic tracheal replacement by a devitalized artery in the dog]. / Zirkulärer intrathorakaler Trachealersatz mit devitalisierter Arterie beim Hund.

Prepared artery (origin: human, calf, dog) with external or internal teflon-spiral-supporting was implanted in dogs (n = 10) as a circular intrathoracic tracheal substitute after cross-resection. Human arteries were rejected (n = 2). Calf arteries (n = 5) and dog arteries (n = 3) were incorporated fibrously. Central granulations appeared. The longest survival time was 36 days.

[The adhesive strength of tissue adhesives]. / Untersuchung der Klebefestigkeit von Gewebekleber.

Twenty concentrates were produced of the blood from voluntary healthy donors by a method for producing autologous tissue adhesive. Their adhesive strength were tested by means of Thermo-fleece-specimens. A review was elaborated about the kinetics and the testing of the fibrin fixation. Methodical faults were found out. Possible ways for improving autologous adhesive variants and for producing homologue tissue adhesive are shown.