SELECTED ABSTRACTS (continued from ETRS Bulletin 14.1+2)

STEM CELLS FROM ADIPOSE TISSUE ALLOW CHALLENGING NEW CONCEPTS FOR SPINAL FUSION

M.N. Helder, J. Klein-Nulend and P.I.J.M. Wuisman.

Dept of Orthopedics, VU University Medical Center, and Dept of Oral Cell Biology, Academic Center of Dentistry Amsterdam, Amsterdam, The Netherlands

Background

Mesenchymal stem cells from adipose tissue (ASCs) can be harvested with minimal patient discomfort with high yields (up to 105/gram tissue), thus eliminating the need for culture expansion prior to implantation. This allows one-step surgical procedures for spinal fusion where harvesting,differentiation induction, scaffold seeding, and implantation can be performed within several hours and within the operational theatre.

Aim:

Development of a one-step surgical procedure in a goat spinal fusion model.

Methods:

Goat and human adipose tissue (AT) were obtained by resection or liposuction. The stromal vascular fraction (SVF) was isolated from the AT using established procedures. ASC content was determined using limiting dilution, CFU-F assays and FACS analysis. Differentiation induction of SVF was accomplished by 15-min stimulation with 10 ng/ml of rhBMP-2 (Control: medium only). Efficacy was verified in vitro by prolonged culturing of stimulated cells for four days in ‘plain’ medium followed by analysis of osteogenic markers (runx-2 and osteopontin gene expression, alkaline phosphatase (ALP) activity).

ASCs were subsequently seeded on calcium phosphate (BCP®) granules. ASC distribution was analyzed by fluorescence and scanning electron microscopy. Finally, the PLDLA cages containing cell-seeded BCP® were used for spinal fusion in the goat. After 28 days, fusion was evaluated using X-rays and histology.

Results:

SVF contained ±105 (goat) or 106 (human) nucleated cells/gram AT, of which up to 8% were ASCs. Upon osteogenic stimulation (15 min of 10 ng/ml BMP-2), expression levels were significantly upregulated compared to controls (1.8-fold and 2.3-fold for runx-2 and osteopontin, respectively;40-fold for ALP activity). Histology/SEM analysis of cellseeded BCP® showed even distribution, and spreading of cells within 30 min. Finally, analysis of the ASC-filled cages after 28 days in vivo revealed both active bone formation (high ALP activity, bone deposition) and remodeling (high number of osteoclasts).

Discussion:

The one-step surgical procedure appears to be feasible, and can be performed within 2-3 hours. Studies with a longer follow-up (3, 6 months) are currently being performed. Although illustrated for spinal fusion, the concept can easily be adapted for other purposes, such as stem cell-directed regeneration of degenerated discs, and other applications.

A NEW APPROACH IN BONE REPAIR BY USING A COMBINATION OF BIPHASIC BIO-CERAMICS, HUMAN MESENCHYMAL STEM CELLS AND PLATELET-DERIVED GLUE

Lataillade Jean-Jacques, MD, Ph.D,

Centre de Transfusion Sanguine des Armées, Unité de Thérapie Cellulaire, BP410, 92141 Clamart Cedex

The Percy French Army Hospital has to treat an important number of patients presenting osteo-articular lesions in which the lost of bone substance is complicated by septic problems. The seriousness and the size of these bone defects, rendering the bone auto graft strategy unsuitable, led us to propose a new stem cell therapy approach.

Progress in tissue engineering makes conceivable the development of macroporous ceramic bone substitutes combined to Mesenchymal Stem Cells (MSC) for filling in the bone defects. Therefore, we proposed a new approach for the reconstruction of critical size bone defects using hybrid implants containing MSC loaded onto HA/TCP ceramics. The novelty of our strategy consisted to combine with these hybrid biomaterials a Platelet-derived Glue (PG) mixture. This PG mixture conferred to the implant many advantages in terms of physiological growths factors release, adhesiveness, malleability and haemostasis.

For this purpose, we have set up two animal models:

1. A mice orthotopic implantation model in which, we have compared the biocompatibility and functionality of MSC versus osteoblastic precursors loaded on HA/TCP bio-ceramics (CERAVER®), in presence or not of PG.
2. A non human primate model of long bone defect in which an ulna critical size defect was performed and stabilized by an internal osteosynthesis. The bone repair was evaluated by local implantation of HA/TCP bio-ceramic loaded by MSC associated with PG.

Our results showed that MSC and osteoprogenitors were able to proliferate at the surface and within the bioceramic. Furthermore, in vitro and in vivo experiments demonstrated that osteogenic differentiation of MSC was improved when PG was associated to the hybrid implant. Osteoblastic precursors appeared to be better candidates than MSC regarding their long term in vivo survival in mice. Their role in critical size bone defect repair has to be evaluated in the non human primate model. Moreover, the PG could be a powerful tool for other tissue regeneration applications.

PREPARING THE WOUND BED FOR FASTER HEALING

G. N. Jukema,

Leiden, The Netherlands

Vacuum-assisted closure (V.A.C. Therapy) uses two distinct types of foams, with different physical characteristics: the black polyurethane (PU) foam and the white polyvinyl alcohol (PVA) foam. This prospective, randomized study evaluates the response of cutaneous blood flow (CBF) in healthy intact forearm skin tovarying V.A.C. Therapy negative pressures and both foam types. Continuous negative pressure was used in the range of 25-500 mm Hg. Skin blood flow as easured with noninvasive laser Doppler probes incorporated into the foam. Significant increase in CBF was found with both foams up to negative pressure of 300 mm Hg, withover 5-fold increase (mean: 5.57; SD: 3.32) with the PU foam and nearly 3-fold increase (mean: 2.87; SD: 1.29) with the PVA foam. Comparison of blood flow at baseline and at a negative pressure of 300 mm Hg showed a statistically significant difference (P < 0.001). No decrease in blood flow below baseline was observed during the experiment

STEM CELLS IN DERMATOLOGY

M. Fimiani, E. Pianigiani,

Dept. of Dermatology, University of Siena

The clinical use of adult and/or embryonic stem cells is emerging as a novel, ambitious therapeutic option in tissue repair and regeneration. In fact, the capacity of adult epithelial stem cells in in vitro regeneration of skin and epithelial tissues is well known since mid seventies and led to a great improvement in the therapy of severe burns, vitiligo, giant melanocytic naevi, skin ulcers and photoaging.

Recently, on the basis of clinical experience in transplantation of organ and haemopoietic cells, relevant data concerning stem cell biological and physiological characteristics, were achieved. These studies allowed a better definition of potential clinical applications and trials of stem cells.

Nowadays, stem cell glossary (niche, homing, cell plasticity, cell fusion, etc.) belongs to current medical terminology. Scientific advantages and potential medical use of adult stem cells have received considerable attention, encouraging stem cell applications in tissue regeneration/repair and immunology.

In recent years, research on factors which guide stem cell differentiation into several types of adult cells, morphologically and functionally differentiated, represents an argument of outstanding importance. It was demonstrated that the environment where stem cells are grown play a crucial role in the differentiation process, besides growth factors. Therefore, research on development of three-dimensional carriers allowing proliferation and differentiation of stem cells, are of great importance as therapeutic options in the treatment of skin loss and leg ulcers.

WHAT IS THE EVIDENCE BASE FOR VAC THERAPY

Paul Banwell,

East Grinstead, UK

The cornerstone of wound-care algorithms in surgical practice has been the reconstructive ladder. Wounds may be allowed to heal by granulation tissue or covered with skin grafts, or even local or distant tissues, using pedicled flaps or microvascular free tissue transfer. However, incorporation of topical negative pressure therapy (TNP - commercially known as V.A.C.® Therapy, KCI, Kiddlington Oxon, UK) into wound management programmes has now enhanced this philosophy. This therapy has extended the indications for simpler surgical techniques in elderly and neurologically-deficient patients and has made it easier to manage complex trauma. An expanding evidence base suggests it should become a mandatory part of the armamentarium of surgeons, nurses and all clinicians involved in wound care. This short talk will aim to review and summarize the current evidence base on V.A.C.® Therapy in terms of physiological and biochemic effects and the associated clinical advantages.

HUMAN FIBROBLASTS ON BIOACTIVE MATERIALS: BEHAVIOUR AND FUNCTION

Bernardini N.¹, D'Alessandro D.¹, Moscato S.¹, Cascone M.G.², Lazzeri L.² and Dolfi A.¹

1. Department of Human Morphology and Applied Biology;
2. Department of Chemical Engineering;

University of Pisa

Tissue regeneration with autologous cell transplantation is one of the most important goals in clinical research. In recent years many synthetic materials have been developed as scaffolds and many procedures for the surface modification of these materials have been applied by using biological molecules to overcome the difficulties of cells adhering to and proliferating on synthetic matrices. Thus, biological research is focusing on the evaluation of morphological features of cells seeded onto bioactive materials. The aim was to investigate the behaviour of human fibroblasts cultured on bioactive materials.

Methods:

Fibroblasts were isolated from human normal gingival mucosa and yellow ligament samples. The normal fibroblast phenotype of the isolated cells was immunohistochemically ascertained. The isolated fibroblasts were seeded on plastic, Matri-cell substrate, rich in basal lamina molecules, or PVA-gelatin sponges, that are highly biocompatible and partially biodegradable scaffolds. Various extracellular matrix molecules (EMMs) were evaluated by histochemical and immunohistochemical techniques: glysosoaminoglycans (GAG) and glycoproteins were evaluated by the Alcian blu method and PAS reaction, respectively; type I collagen, fibronectin, and laminin were revealed by immunohistochemistry.

Results and Conclusions:

Typical fibroblast-like phenotype was observed in the isolated human cells which were able to grow on plastic and produce EMMs. No morphological differences were found between the cells grown bi-dimensionally (plastic or Matricell) or three-dimensionally (PVA-gelatin sponges). When cultured on bioactive materials, the cells maintained the immunohistochemical markers of fibroblastic phenotype as well as the ability to produce appreciable amounts of EMM. Thus, we can conclude that:

1. primary cultures obtained from human gingiva and yellow ligament are fibroblasts;
2. on the tested bioactive materials these cells are able to adhere, proliferate, produce EMM maintaining fibroblastic features;
3. there are no differences in morphological or immunohistochemical behaviour on PVA-gelatin sponges of fibroblasts isolated from gingival or ligament tissues;
4. the tested PVA-gelatin sponges are suitable for use as scaffolds for three-dimensional adhesion and growth of human fibroblasts.

THREE-DIMENSIONAL IN VITRO ORGAN CULTURE MODEL OF THE POTENTIAL APPLICATION IN PEDIATRIC SURGERY

G. Tanda, C. Spinelli.

Department of Pediatric Surgery, University of Pisa, Italy (Direttore: Prof. Claudio Spinelli)

Research into methods of selection and growth of epithelial basal cells is important for its potential use in pediatric surgery.

The 3-D organ culture model is an advanced technique in the culture of skin/organs in vitro which allows the organ to maintain its tissue architecture and cellular heterogeneity the capacity of the cells to reply to various stimuli of the micro-environment in culture over a long period. To this end we used the peritoneal vaginal duct (DPV) removed/extracted in the operating theatre from 20 children undergoing surgery for inguinal hernia or Hydrocele. Once the fragments of DPV have spent 4-5 hours in the decontaminated solution ( an antibiotic/antimicotic solution, 1,25 X) they are cut up and placed on collagen sponge. Each collagen sponge is soaked in Epilife culture enriched with FBS at 10%, and are transferred along with the fragments of DPV into a well of tissue culture plate. The culture is kept in a humidified incubator at 37°C, in an atmosphere made up of 5% CO2. The culture medium (means) is changed every two days. The fragments are kept in culture for 7 days (T1), 14 days (T2), and 21 days (T3). The samples at T0, T1, T2, T3, and their relative sponges, were examined by means of careful histological and immunohistochemical analysis. The markers used in this studying were: anti Ki-67 as a cycling cell marker, anti CK19 as precursor epithelial marker. The histological test showed that before the culture the normal DPV was made up of a epithelium monostratified which was lying on a basal lamina only barely evident. The stroma mainly vascularized, is rich of fibrillar matrix and cells. The cytokeratins AE1-AE3 and in particular cytokeratin19 were mainly expressed in the epithelium. These cells also showed the cycling cell marker Ki-67 confirming the role of reserve of the basal cell. The vimentin was localized into stroma and endothelia. Seven days into the culture the histological test showed a loss of continuity of the epithel. After 14 days the histological test regeneration of the epithelium which expressed (AE1-AE3+ e CK 19+) similar to that at T0 even if the fraction of positive cells increases for vimentin. As time passes the epithelium covered the whole fragment; we were noted the presence of cells invading the sponge. The stroma and endotelia were well preserved. After 21 days the cytokeratins AE1-AE3 were still expressed in the epithelium while the cytokeratin 19 was reduced. Studies carried out on the 3D organ culture model of DPV have shown that it is possibile to keep even extremely delicate epithelia like the epithelium monostratified of the DPV in culture over a medium or long period of time. The results lead us to believe that the DPV can, in the near future, be used as homologous tissue which recreated correctly in the 3D organ culture model, can be used to correct congenital anomalies in children.

THE CLINICAL IMPACT OF WOUND INFECTION. A SINGLE CENTER ANALYSIS ON 1000 PATIENTS WITH DIABETIC FOOT ULCERS

S. Coerper, S. Beckert and A. Koenigsrainer.

Dept. of General Surgery, University Clinic, Tübingen, Germany.

Introduction:

There is agreement that diabetic foot infection represents a high risk for limb and life of diabetic patients. We investigated the course of healing in respect to wound infection and evaluated factors that might favor wound infection in diabetic patients.

Patients and Methods:

Patients were treated according to a standardized wound care protocol. In total, 1000 patients were included in this study. Mean age was 68+11 years. Follow-up was documented within a special wound documentation system. Wound infection was defined by clinical criteria. We distinguished primary wound infection at the first visit (Primary WI) and postoperative wound infection (Postop. WI). The probability for healing or amputation was calculated by Kaplan Meier analysis and differences were calculated with the log rank test. Impact factors for the incidence of wound infection were calculated by the Chi-Quadrat test. Data is given as mean+SD.

Results:

Mean time of follow-up was 110+111 days and wound history 142+674 days (21%>120 days). Sharp debridement was performed in 100%, bone resection in 13.6%, minor amputation in 9.9% and major amputation in 2.6%. There were 35% recurrent ulcers, 35.6% toe, 64.4% foot ulcers, in 26.9% bone was exposed, in 34.6% pedal pulses were not palpable and mean ulcer size was 4.76+12.16cm² (48% >1cm²). Primary WI (35%): Probability for healing was not affected (p = 0.53). There was an increased probability for minor amputation (p = 0.0001) but not for major amputation (p = 0.40). Infection rate was increased for ulcers with bone involvement (p = 0.001), toe ulcers (p = 0.0001), ischemia (p = 0.0001) and larger ulcers (p = 0.007). Postop. WI (20%): Probability for healing was decreased (p = 0.0001). There was no difference in respect to minor (p = 0.10) or major amputation (p = 0.31). Infection rate was increased for toe ulcers (p = 0.002), large (p = 0.001), recurrent (p = 0.029) and multiple (p = 0.027) ulcers.

Conclusion:

According to our data, the probability for major amputation was not increased by neither initial nor postop. WI. The probability of healing was significantly affected exclusively by postop. but not initial wound infection. We conclude that initial and postop. WI have a different impact factors on healing.

THE EFFECT OF SUPRASORB X ON HARD TO HEAL ULCERS. A NON CONTROLLED PROSPECTIVE ANALYSIS ON 96 PATIENTS

S. Coerper, S. Beckert, G. Deutschle, C. Halm-Nill and A. Koenigsrainer.

Dept. of General Surgery, University Clinic, Tübingen, Germany.

Introduction:

Suprasorb® X is a sterile product composed of cellulose, water, and 0.085% chlorhexidine gluconate. We report about the success of the treatment with Suprasorb® X (earlier: Xcell) on patients with hard to heal ulcers.

Methods:

Treatment of patients with chronic wounds is performed in an interdisciplinary wound care centre according to a comprehensive wound care protocol. Follow up was documented within a special documentation system. For analysis in patients with multiple ulcers the larger ulcer was defined as a primary ulcer. Pain was defined according to a scale (1-10) and assessed the last visit before and 3 weeks after Suprasorb® X therapy. Data was analysed using SPSS and is given as median and range. Healing rates were calculated with the log rank test because of different observation periods.

Results:

We treated 39 patients with venous, 24 with ischemic, 19 with diabetic and 14 with ulcers of different ethiology. There were 40 patients with multiple ulcers, 23 with circumferential ulcers and wound size of the other 73 patients was 11(1-263cm²). In 33% there was bone involvement and in 29% initial wound infection evident. The treatment time of unsuccessful therapy before was 178 (6-2052) days, treatment time with Suprasorb® X was 119 (7-810)days. There was no intolerance or allergy to Suprasorb® X and in 43% of the patients pain reduction was evident already one visit after start of Suprasorb® X therapy. The over all healing rate was 52% within the time of treatment. The healing rate of diabetic ulcers was 60%, of venous ulcers 43% and of ischemic ulcers 5%.

Conclusion:

These patients had hard-to-heal ulcers, demonstrated by base line characteristics and the time duration of unsuccessful therapy before. However, there was an over all healing rate of more than 50%. Ischemic ulcers did not respond to Suprasorb® X therapy. In contrast to other wound dressings Suprasorb® X was well tolerated, no allergy or skin irritation was found and an obvious pain reduction was found. Prospective studies are underway to evaluate the impact of Suprasorb® X.

PLATELET-RICH FIBRIN (PRF) AND ITS PROLONGED BENEFICIAL EFFECTS ON FIBROBLAST ACTIVITIES IN VITRO

Lundquist R, Danielsen P, Jorgensen LN, Karlsmark T and Ågren MS.

Department of Surgery K, Bispebjerg Hospital, Copenhagen University Hospital, Copenhagen, Denmark.

Aims

Platelets contain potent growth factors and are used topically in various carrier systems in the treatment of acute and chronic wounds. Including platelets in the autologous fibrin prepared at the bedside is a novel formulation (Vivostat, Vivolution, Denmark). The combination of platelets and fibrin is believed to increase stability and prolong bioactivity of the included growth factors in highly proteolytic chronic wound milieu. The aims of these studies were to assess the effect of the novel topical autologous platelet-rich fibrin (PRF) preparation on fibroblasts cultured in vitro. Also, stability studies on PDGF-AB, the most abundant growth factor in human platelets, in the PRF were carried out.

Methods:

The effect of PRF on fibroblast proliferation was assessed in confluent and growth-arrested adult human dermal fibroblasts (Cambrex). The fibroblasts were treated with fibroblast basal medium (FBM) containing 2% fetal calf serum (FCS) in the absence or presence of pre-formed PRF-clots (n = 12) or of 10 ng/ml platelet derived growth factor-BB (n = 12, PDGF-BB) for 24 hours in 96-well culture plates. BrdU incorporation was measured during the final 2-hour incubation period using a cell proliferation ELISA kit (Roche). To assess the effects on prolonged cell growth an assay starting with low cell numbers (2,000 fibroblasts in FBM + 2% FCS/well) in 96-well culture plate was applied. Cell numbers were followed for 96 hours at 24-hour intervals by ATP-detection using an ATP-standard (ViaLight, Cambrex). Stability studies on PDGF-AB in PRF were performed by the addition of specific proteases or wound fluid from chronic venous leg ulcers. Levels of intact PDGF-AB after the various treatments was detected by specific ELISA assay.

Results:

The in vitro assays showed significant increased proliferation and cell growth with PRF (p < 0.001) and PDGF-BB (p < 0.001) compared with control (FBM + 2% FCS). Results from growth factor stability studies using both specific proteases and chronic wound fluid will be presented.

Conclusions:

The platelet-rich fibrin (PRF) exhibited fibrogenic properties in vitro. Besides the autologous origin and easy preparation and application at the bedside of PRF, the combination of fibrin and platelets leads to the administration of growth factors protected from the proteolytic wound environment.

NEGATIVE PRESSURE WOUND THERAPY ON THE PRIMARY CLOSED WOUND FOR PREVENTION OF WOUND INFECTION: A PILOT STUDY

N.M. Graafland,¹ M. Hogervorst,¹ M.S. Timmers,² and G.N. Jukema.²

1. Dept of Surgery, Medical Center Haaglanden, The Hague.
2. Dept of Surgery, Section of Traumatology, Leiden University Medical Center, Leiden, The Netherlands.

Background:

Negative pressure wound therapy (NPWT) by means of the vacuum assisted closure (V.A.C.) system is widely used in various types of wounds. In spite of the fact that the proposed working mechanisms of healing could also be beneficial in closed wounds, they are until now terra incognita.

Aims:

To study the feasibility of V.A.C. therapy on the closed wound, with the emphasis of detection of adverse effects and prevention of wound infection.

Methods:

All primary closed wounds that were prone for infection after operation were eligible for study inclusion. A polyvinyl alcohol foam which overlapped the incision with 3 centimetres on each side with negative pressure of 100 mmHg was applied either directly post-operative or during admission when an impaired wound healing was imminent. Exclusion criteria were: pus retention, sepsis or no V.A.C. pump available.

Result:

A total of 21 patients (range, 22-96; mean, 67 years) were treated with V.A.C. therapy on closed wounds; hemiarthroplasty 9, bowel surgery 5, lower limb amputation 4, osteosynthesis with elevated infection risk 3. Mean duration of therapy was 3.9 days (range, 1-7), with average 0.9 foam changes (range, 0-3), all performed at bed side. There were 7 minor skin complications. All healed spontaneously after V.A.C. therapy In one patient with imminent infection, therapy was stopped prematurely because of skin irritation and a wound infection occurred. In two patients a wound healing occurred after V.A.C. therapy.

Conclusions:

A minority of patients experienced temporary, adverse effects of the skin. These adverse effects were not related to duration of therapy and were no reason to end V.A.C. therapy prematurely. In these patients with high risk of infection, V.A.C. therapy seems to prevent and possibly even treat infection in closed wounds. This has opened the way for further investigations to compare wound outcome with conventional therapy.

THE EFFECTS OF VARYING DEGREES OF PRESSURE DELIVERED BY NEGATIVE PRESSURE WOUND THERAPY ON SKIN PERFUSION

Timmers M.S.¹ Le Cessie S.³ Banwell P.² and Jukema G.N.¹

1. Section of Traumatology, Dept of Surgery Leiden University Medical Center, Leiden, The Netherlands.
2. Odstock Centre for Burns, Plastic & Reconstructive Surgery, Salisbury District Hospital, Salisbury, UK.
3. Dept of Medical Statistics Leiden University Medical Center, Leiden, The Netherlands.

Aims:

The aim of this study was to investigate the effects of varying degrees of pressure by negative pressure wound therapy on skin perfusion with use of two different foam types.

Materials and methods:

This prospective, randomised study evaluates the response of cutaneous blood flow(CBF) in healthy intact forearm skin to varying negative pressures and use of polyvinyl alcohol(PVA) and polyurethane(PU) foam. Continuous negative pressure was used in the range of 25-500 mmHg. Skin blood flow was measured with non-invasive laser Doppler probes incorporated into the foam.

Results:

Statistically significant increase in CBF was found with PU and PVA foam up to negative pressure of 300 mmHg, with over five-fold increase (mean: 5.57; SD: 3.32) with the PU foam and nearly three-fold increase (mean: 2.87; SD: 1.29) with the PVA foam. Comparison of blood flow at baseline and at a negative pressure of 300 mmHg showed a statistically significant difference (p < 0.001). No decrease in blood flow below baseline was observed during the experiments.

Conclusion/Discussion:

This study, with its statistically significant rise in CBF with both the PU and PVA foam up to a pressure of 300 mmHg, in combination with our clinical data suggest that the pressure of 125 mmHg, which is the standard negative pressure used for negative pressure wound therapy might not be the optimal pressure for enhancing blood flow in intact human skin. A relation between different negative pressure levels on foam dressings and cutaneous blood flow could be established.