Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety

Since 1987, keratinocytes have been cultured at the Queen Astrid Military Hospital. These keratinocytes have been used routinely as auto and allografts on more than 1,000 patients, primarily to accelerate the healing of burns and chronic wounds. Initially the method of Rheinwald and Green was used to prepare cultured epithelial autografts, starting from skin samples from burn patients and using animal-derived feeder layers and media containing animal-derived products. More recently we systematically optimised our production system to accommodate scientific advances and legal changes. An important step was the removal of the mouse fibroblast feeder layer from the cell culture system. Thereafter we introduced neonatal foreskin keratinocytes (NFK) as source of cultured epithelial allografts, which significantly increased the consistency and the reliability of our cell production. NFK master and working cell banks were established, which were extensively screened and characterised. An ISO 9001 certified Quality Management System (QMS) governs all aspects of testing, validation and traceability. Finally, as far as possible, animal components were systematically removed from the cell culture environment. Today, quality controlled allograft production batches are routine and, due to efficient cryopreservation, stocks are created for off-the-shelf use. These optimisations have significantly increased the performance, usability, quality and safety of our allografts. This paper describes, in detail, our current cryopreserved allograft production process

Glycerol treatment as recovery procedure for cryopreserved human skin allografts positive for bacteria and fungi

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts

Long-term results after excision haemorrhoidectomy versus stapled haemorrhoidopexy for prolapsing haemorrhoids a belgian prospective randomized trial

Purpose: To compare the postoperative evolution and the long-term efficacy after stapled haemorrhoidopexy (PPH) and Milligan-Morgan haemorrhoidectomy (MM). Methods: In a prospective randomized study, 40 patients requiring surgical treatment for prolapsing haemorrhoids grade II or III were assigned to either MM or PPH (20 each). Postoperative pain, wound healing were evaluated, as well as anal pressures and sphincter anatomy. Mean follow-up is 46 months. Results: Postoperative pain at rest and during defecation was less important after PPH if no resection of external piles or skin tags was associated (P < 0.0001). Healing time was shorter after PPH (P < 0.0001). Endoanal ultrasound remained unchanged postoperatively. Resting and squeeze pressures decreased after MM, but not after PPH (P < 0.01). After a mean follow-up of 46 months (12-56), persistent or recurrent symptoms, mostly mild and temporary, were observed after both MM and PPH, in 7 and 11 patients respectively (NS). After PPH, five patients (25%) complained of recurrent external swelling and/or prolapse (P = 0.047 vs. MM) requiring redo surgery in four of them, after 10, 13, 14 and 21 months. No redo-surgery was required after MM. Long term patient satisfaction after PPH was not better than after MM. Conclusions: Postoperative pain is less important after PPH. This advantage disappears if any resection is associated with the stapling. At medium to long-term follow-up, PPH seems to carry a higher risk of symptomatic external haemorrhoidal disease, needing further surgery.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted