A novel DNA profiling application for the monitoring of cross-contamination in autologous chondrocyte implantation.

BACKGROUND: Autologous chondrocyte implantation (ACI) is a cell-based treatment that can be used to regenerate chondral defects. European legislation specifically classifies such produced chondrocytes as "medicinal for advanced cell therapy" that have to be manufactured in pharmaceutical factories according to specific rules, named Good Manufacturing Practices (GMPs). One main requirement of cell manipulation in advanced therapy is to prevent the risk of any contamination. AIM: The aim of this study was to verify if chondrocyte cultures suitable for ACI were free of cross-contamination by means of DNA profiling techniques. MATERIALS AND METHODS: Cell cultures were carried on in a Hospital Cell Factory in compliance with European current Good Manufacturing Practices. DNA profiling, by means of Short Tandem Repeats and miniShort Tandem Repeats analyses, was performed on expanded chondrocytes and their related control blood samples. Mitochondrial DNA was analysed to further confirm the results and to evaluate possible mutations occurred in the samples. RESULTS: Our findings demonstrated the absence of cross-contamination between chondrocyte cultures and, thus, their identity maintenance until the end of the manipulation. CONCLUSIONS: DNA profiling technique can be a suitable test for quality control not only for chondrocyte manipulation, but for cell therapy in general.

In vivo study on the healing of bone defects treated with bone marrow stromal cells, platelet-rich plasma, and freeze-dried bone allografts, alone and in combination.

The repair of confined trabecular bone defects in rabbits treated by autologous bone marrow stromal cells (BMSC), platelet-rich plasma (PRP), freeze-dried bone allografts (FDBA) alone and in combination (BMSC + PRP; FDBA + BMSC; FDBA + PRP; FDBA + PRP + BMSC) was compared. A critical size defect was created in the distal part of the femurs of 48 adult rabbits. Histology and histomorphometry were used in the evaluation of healing at 2, 4, and 12 weeks after surgery. The healing rate (%) was calculated by measuring the residual bone defect area. Architecture of the newly formed bone was compared with that of bone at the same distal femur area of healthy rabbits. The defect healing rate was higher in PRP + BMSC, FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC treatments, while lower values were achieved with PRP treatment at all experimental times. The highest bone-healing rate at 2 weeks was achieved with FDBA + PRP + BMSC treatment, which resulted significantly different from PRP (p < 0.05) and BMSC (p < 0.05) treatments. At 4 weeks, the bone-healing rate increased except for PRP treatment. Finally, the bone-healing rate of FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC was significantly higher than that of PRP at 12 weeks (p < 0.05). At 12 weeks, significant differences still existed between PRP, BMSC, and FDBA groups and normal bone (p < 0.05). These results showed that the combination of FDBA, BMSC and PRP permitted an acceleration in bone healing and bone remodeling processes.

Evaluation of bone healing enhancement by lyophilized bone grafts supplemented with platelet gel: a standardized methodology in patients with tibial osteotomy for genu varus.

Orthopedic practice may be adversely affected by an inadequate bone repair that might compromise the success of surgery. In recent years, new approaches have been sought to improve bone healing by accelerating the rate of new bone formation and the maturation of the matrix. There is currently great interest in procedures involving the use of platelet gel (PG) to improve tissue healing, with satisfactory results both in vitro and in maxillofacial surgery. Otherwise, to our knowledge, only a preliminary clinical study was undertaken in the orthopedic field [Kitoh et al., Bone 2004;35:892-898] and the efficacy of PG is still controversial. Our paper focuses on the effect on bone regeneration by adding PG to lyophilized bone chips used for orthopedic applications. The clinical model and the laboratory methodology were standardized. As a clinical model, we employed the first series of patients of a randomized case-control study undergoing high tibial osteotomy (HTO) for genu varus. Ten subjects were enrolled: in 5 patients lyophilized bone chips supplemented with PG were inserted during tibial osteotomy (group A); 5 patients were used as a control (group B) and lyophilized bone chips without gel were applied. Forty-five days after surgery, computed tomography scan guided biopsies of grafted areas were obtained and the bone maturation was evaluated by a standardized methodology: the osteogenic and angiogenic processes were semi-quantitatively characterized by using histomorphometry, and the mineral component of the lyophilized and host bone was analyzed by using X-ray diffraction technique with sample microfocusing and microradiography. Lyophilized bone with PG seems to accelerate the healing process, as shown by new vessel formation and deposition of newly formed bone, with no evidence of inflammatory cell infiltrate, when compared with lyophilized bone without gel. On the contrary, lyophilized bone undergo a resorption process, and a fibrous tissue often fills the spaces between chips. A histiocytic/giant-cell reaction is sometimes present. Otherwise, no differences have been found concerning microstructure. Our findings show the reliability of the methodology used to monitor early bone repair. The completion of the study and the evaluation of the ultimate clinical outcome are necessary in order to verify PG in vivo effects in orthopedic surgery.

Blood vessel formation after soft-tissue implantation of hyaluronan-based hydrogel supplemented with copper ions.

The possibility of ameliorating bone healing of implanted bone allografts is a field of great interest. Early vascular invasion is a key factor in bone allograft incorporation. It is well known that copper ions (Cu2+) show a proangiogenic action favouring the development of new vessels. In this work a hyaluronan based 50% hydrogel (Hyal-50%) was enriched with (Cu2+) and its proangiogenic activity was evaluated. Fifteen Sprague Dawley female rats were submitted to the subcutaneous implantation of Hyal-50%, freeze-dried bone allografts, Hyal-50%-Cu2+, freeze-dried bone allografts plus Hyal-50% and freeze-dried bone allografts plus Hyal-50%-Cu2+. One month later, histomorphometric analysis evidenced the presence of a fibrous-reactive capsule around all specimens showing significant differences among groups (p<0.0005). The highest thickness of the fibrous capsule was found around the freeze-dried bone implants (p<0.05); as well as the Hyal-50%-Cu2+ plus freeze-dried bone (15.2%, p<0.05) and Hyal 50% plus freeze-dried bone (21.4%, p<0.0005) implants showed a significant higher thickness compared with Hyal 50% and Hyal-50%-Cu2+. Statistical analysis showed a significant (p<0.01) higher vascular density in Hyal- 50%-Cu2+ and Hyal-50%-Cu2+ plus freeze-dried bone group when compared to other groups. The present preliminary results suggest the advantages offered by the combined use of a well-known biocompatible and tissue healing promoting material (Hyal-50%) and a new technique that consists of stimulating tissue vascularization using Cu2+ and that bone allograft incorporation may benefit from this technology.

Hemapheresis and plasma plan.

The Italian Authorities have recently presented the National Blood and Plasma plan for the period 1993-1995. One of the main topics is the regional and national plasma-derivatives self-sufficiency by whole blood fractionated plasma increase and donor plasmapheresis activation. Plasmapheresis will follow efficiency and efficacy criteria.

Short and long-term clinical effects of plasma-exchange in 33 cases of myasthenia gravis.

The authors examine the results of plasma exchange (PE) treatment in 33 patients with myasthenia gravis, who underwent a total of 49 cycles of treatment. On the basis of clinical response, evaluated in relation to a modification of the functional state and to the duration of improvement after PE, we selected a group of 20 responsive patients ("responders" = 60.6%) and 13 non-responsive patients ("non-responders" = 39.4%). Positive effects, some only short-lasting, were observed in 24 patients (72.7%) after 32 cycles of a total of 49 cycles (65.3%). Out of the 11 most critical patients, 7 (63.6%) were considered responders, given the importance and duration of the improvement they presented, while 2 other patients had clear but short-lasting improvements; in these 9 patients, the positive effects amounted to 81.2%. Factors presumably responsible for therapeutic failure in the non-responders are discussed.

Donor plasmapheresis: a comparative study using four different types of equipment.

From September 1985 to May 1986, 106 healthy volunteers underwent automated plasmapheresis in our Institution (CT-AVIS, PAVIA) using four different types of equipment. The aim of this study was to verify the feasibility of harvesting plasma from a single donor and to ascertain: the acceptability of the procedure to donors; the speed, economy and safety of the different types of equipment; the quality of the plasma. The equipment employed in this study was: PCS (Plasma Collection System-Haemonetics) FILTRA (Dideco) PLASMAPUR (Organon Teknika). AUTOPHERESIS CTM-(Hemascience). All four used a single-needle technique. Procedures were well tolerated by donors; no significant changes were observed in pre- and post-procedure laboratory values.

[Plasmapheresis and survival of patients with myeloma and secondary renal insufficiency]. / Plasmaferesi e sopravvivenza del paziente mielomatoso con insufficienza renale secondaria.

The mortality rate due to renal insufficiency in patients with myelomas was studied and the results obtained compared with figures on patients given plasmapheresis to correct the insufficiency. The results of the comparison confirm the value of the immediate use of plasmapheresis on patients with myelomas. In a large percentage of cases, the technique either completely cured or significantly improved renal dysfunction, thus normalizing the blood and urine situation and improving the patients' chances of survival.

[The role of plasmapheresis in the treatment of primary and secondary cryoglobulinemia]. / Ruolo della plasmaferesi nel trattamento delle crioglobulinemie primarie e secondarie.

The data on monoclonal or mixed cryoglobulinaemia patients admitted to Pavia University's 1st Medical Division since 1970 were examined. Complications included peripheral microangiopathies caused by immune complexes and especially kidney lesions of varying severity caused by immune complexes precipitated in the basal membrane. Although the course of the condition was ameliorated in all patients given immunosuppressive treatment alone, this could not prevent the appearance of progressively worsening kidney conditions. Terminating in severe renal failure these were often complicated by encephalopathies, disturbed microcirculation in the brain and hypertension patients given plasmapheresis at an early stage, at the first sign of renal or brain circulation problems, responded brilliantly with regression of all clinical symptoms. Where plasmapheresis was not given until an advanced stage of the kidney disease, a satisfactory improvement was obtained and the quality and expectancy of life enhanced, even though a total cure was not achieved. Regular cycles of plasmapheresis indubitably protect the patient from irreversible renal or microvascular conditions so that immunosuppressive treatment can effectively control the cryoglobulinaemia.

Bacterial contamination of musculoskeletal allografts.

Based on the indications in the literature of the transmission of highly pathogenic bacteria in musculoskeletal allograft implants, the cultural results for allografts removed and implanted in conditions of asepsis between 1997 and 2000 in BTM were analyzed: 4014 allografts (3117 from a living donor, 897 from a cadaver) and 3479 implants (2191 with allografts from living donors and 1288 with allografts from cadavers). Explanted tissues: these were positive to culture in 292 out of 3117 (9.4%) allografts from living donors; the bacteria isolated showed low pathogenicity. Out of 897 allografts 117 cadaver donors bacteria with low pathogenicity were isolated in 68 (7.6%) and high pathogenicity in 12 (1.3%). Implants: cultures were positive in 116/2191 (5.3%) implants with allografts from living donors and in 55/1288 (4.3%) implants with allografts from cadavers. The bacteria isolated are the same as those shown in the explants. In living donors contamination is similar as regards incidence and type of microorganism to that observed in surgical theatres during routine surgery. Contamination seems to be greater in allografts removed from cadavers who died as a result of trauma, in the presence of positive hemocultures, prolonged catheterization and intubation, explantation of the pelvis and removal of several organs and tissues prior to musculoskeletal allograft.

European quality system for tissue banking.

AIM: The aims of this project were to analyze the factors that influence quality and safety of tissues for transplantation and to develop the method to ensure standards of quality and safety in relation to tissue banking as demanded by European Directive 2004/23/EC and its technical annexes. It is organized in 4 Working Groups, the objectives of each one being focused in a specific area. STANDARDS: The Guide of Recommendations for Tissue Banking is structured into 4 parts: (1) quality systems that apply to tissue banking and general quality system requirements, (2) regulatory framework in Europe, (3) standards available, and (4) recommendations of the fundamental quality and safety keypoints. REGISTRY: This Working Group handled design of a multinational musculoskeletal tissue registry prototype. TRAINING: This Working Group handled design and validation of a specialized training model structured into online and face-to-face courses. The model was improved with suggestions from students, and 100% certification was obtained. AUDIT: The Guide for Auditing Tissue Establishments provides guidance for auditors, a self-assessment questionnaire, and an audit report form. The effectiveness and sustainability of the outputs were assessed. Both guides are useful for experienced tissue establishments and auditors and also for professionals that are starting in the field. The registry prototype proves it is possible to exchange tissues between establishments throughout Europe. The training model has been effective in educating staff and means having professionals with excellent expertise. Member states could adapt/adopt it. The guides should be updated periodically and perhaps a European organization should take responsibility for this and even create a body of auditors.

Enhanced tibial osteotomy healing with use of bone grafts supplemented with platelet gel or platelet gel and bone marrow stromal cells.

BACKGROUND: There is great interest in the use of bone substitutes to improve bone repair. We compared the osteogenic potential of lyophilized bone chips combined with platelet gel, or with platelet gel and bone marrow stromal cells, with that of lyophilized bone chips alone in the healing of a high tibial osteotomy. METHODS: A prospective, randomized, controlled study was performed, and a standardized clinical model was applied. Thirty-three patients undergoing high tibial osteotomy to treat genu varum were enrolled and assigned to three groups. During the osteotomy, lyophilized bone chips with platelet gel were implanted into eleven patients (Group A), lyophilized bone chips with platelet gel and bone marrow stromal cells were implanted in twelve patients (Group B), and lyophilized bone chips without gel were placed in ten patients as controls (Group C). Six weeks after surgery, computed tomography-guided biopsies of the grafted areas were performed and the specimens were analyzed by histomorphometry. Clinical and radiographic evaluation was performed at six weeks, twelve weeks, six months, and one year after surgery. RESULTS: Histomorphometry at six weeks showed significantly increased osteoblasts and osteoid areas in both Group A (p = 0.006 and p = 0.03, respectively) and Group B (p = 0.009 and p = 0.001) in comparison with controls, as well as increased bone apposition on the chips (p = 0.007 and p = 0.001, respectively), which was greater in Group B than in Group A (p < 0.05). Group B showed significantly higher revascularization than the controls (p = 0.004). Radiographs revealed a significantly higher rate of osseointegration in Groups A and B than in the controls at six weeks (p < 0.005 and p < 0.0001, respectively). At the final evaluation at one year, the osseointegration was still better in Groups A and B than in Group C; however, all patients had complete clinical and functional evidence of healing. CONCLUSIONS: Adding a platelet gel or a platelet gel combined with bone marrow stromal cells to lyophilized bone chips increases the osteogenetic potential of the lyophilized bone chips and may be a useful tool in the treatment of patients with massive bone loss.

[Monitoring of lymphocyte classes and subclasses during lymphocyte plasmapheresis]. / Monitoraggo delle classi e sottoclassi linfocitarie in corso di plasmalinfocitoaferesi.

The authors have investigated the immunological picture in one group of patients affected by immune mediated diseases treated with lymphoplasmapheresis. The immunomodulatory effects of the therapy were controversial after a single procedure, paradoxical after a complete cycle of 4 procedures.

Clinical effects of plasmapheresis in acute Guillain-Barré syndrome.

5 consecutive cases of acute GBS were treated by plasmapheresis (PP). Clinical features and results of therapy have been compared with those of 10 consecutive acute GBS cases untreated by PP. PP treatment, applied early during the phase of deterioration, seemed to stop deficit progression, thus eliminating the stationary stage and triggering recovery immediately. Complete recovery seemed to occur comparatively faster than in the untreated group.

Haemostasis during transvesical prostatic adenomectomy. A controlled trial on the effect of drugs with antifibrinolytic and thrombin-like activities.

Blood loss, plasma fibrinolytic activity, prothrombin, P.T.T., and fibrinogen plasma levels were measured in 30 patients subjected to transvesical adenomectomy. These parameters were evaluated during 7 consecutive days after the operation. Ten patients received the antifibrinolytic substance (AMCHA), ten patients received Bothrops Jararaca venom extract, and ten patients served as controls. The results show that the coagulating fraction of Bothrops Jararaca snake venom reduces intraoperative bleeding without influencing the haemostatic balance. Postoperative haematuria could be reduced by antifibrinolytic agents.