A novel system for expansion and delivery of human keratinocytes for the treatment of severe cutaneous injuries using microcarriers and compressed collagen

Cell therapy with autologous or allogeneic keratinocytes applied as a single‐cell suspension is well established in clinical practice in the treatment of severe burn injuries to augment epithelial barrier restoration. Yet, the application of cell sprays can lead to significant cell loss owing to lack of adhesion of cell suspension to the wound bed. The development of a robust and controllable method of transplanting cells onto the wound bed is yet to be established. The ability to control adhesion and distribution of cells by using a cell carrier embedded in a biodegradable scaffold could significantly improve the treatment of cutaneous wounds with keratinocyte cell therapy. Several microcarrier‐based systems for expanding keratinocytes already exist. A new method for expansion of human keratinocytes in a feeder‐free, defined medium system on microcarriers has been developed. The cells retained their basal, proliferative phenotype after rapid expansion in a clinically relevant time‐frame. The cell‐laden microcarriers were further incorporated into collagen scaffolds fabricated by plastic compression. When cultured in vitro, cells continued to proliferate and migrate along the surface of the collagen scaffold. Using an in vitro wound bed model, cells were observed to form mostly single cell layers and in some areas multiple cell layers within 8 days, while retaining their basal, proliferative phenotype, indicating the suitability of this cell transplantation method to improve epithelial barrier restoration. This advanced cell expansion and delivery method for cutaneous cell therapy provides a flexible tool for use in clinical application. Copyright © 2017 John Wiley & Sons, Ltd.     

Evaluation of autologous cultured epithelium as replacement skin after tattoo excision: correlation between skin texture and histological features

BACKGROUND: Cultured epidermal autographs (CEAs) are currently used as a coverage treatment for burn wounds, for disfiguring burn scars involving depigmentation and in restoring the elasticity of the skin. The advantage of CEAs is that epidermal sheets prepared from small skin pieces can be enlarged sufficiently to cover large burn areas. OBJECTIVES: We examined the correlation between recovery of skin texture, and elastic fibre formation and keratinocyte differentiation (assessed by immunohistochemistry) in CEAs used as replacement skin after tattoo excision in a Japanese patient. METHODS: The tattooed skin was excised down to the deep dermal layer and then CEA was transplanted onto the patient. The skin textures were evaluated by taking replicas of the skin surface, and histological changes of filaggrin, transglutaminase, involucrin, fibrillin and elastin in the autograft skin were examined by immunohistochemistry. RESULTS: The skin texture improved with time after grafting the CEA, and appeared similar to that of normal skin at 39 months. Among keratinocyte differentiation markers, filaggrin recovered to a normal pattern at around 6 months, and transglutaminase did so at 39 months, whereas involucrin expression remained abnormal at 39 months. Fibrillin expression appeared similar to that of normal skin by 39 months, except for sparse candelabra-like structures of short fibres. Elastin expression remained at a low level throughout. CONCLUSIONS: Our results show that the recovery of skin texture after application of CEAs following tattoo excision is associated with the normalization of epidermal differentiation markers, except involucrin, and with the regeneration of elastic fibres in the dermis.     

The host environment determines strain-specific differences in the timing of skeletal muscle regeneration: cross-transplantation studies between SJL/J and BALB/c mice

The difference in the timing of the regeneration process of skeletal muscle between SJL/J and BALB/c mice was investigated using grafts of whole skeletal muscle (both autografts and allografts). Histological, autoradiographic and immunohistochemical techniques were used in the investigation. Infiltration of leucocytes into autografts, numbers of desmin-positive myogenic cells and myotube formation were all more advanced in the SJL/J compared with BALB/c mice. Furthermore, autoradiographic evidence showed that myoblasts in the SJL/J autografts were synthesising DNA 12 h earlier than myoblasts in BALB/c autografts. In allografts, where SJL/J host mice received BALB/c grafts, and vice versa, leucocyte infiltration and myotube formation occurred earlier in the BALB/c muscles grafted into SJL/J hosts, than in the reverse situation with BALB/c hosts. The results show that, at least for whole muscle grafts, it is the host environment which determines the speed and outcome of the regenerative process.     

Cartilage repair with osteochondral autografts in sheep: Effect of biophysical stimulation with pulsed electromagnetic fields

The effect of pulsed electromagnetic fields (PEMFs) on the integration of osteochondral autografts was evaluated in sheep. After osteochondral grafts were performed, the animals were treated with PEMFs for 6 h/day or sham‐treated. Six animals were sacrificed at 1 month. Fourteen animals were treated for 2 months and sacrificed at 6 months. At 1 month, the osteogenic activity at the transplant–host subchondral bone interface was increased in PEMF‐treated animals compared to controls. Articular cartilage was healthy in controls and stimulated animals. At 6 months, complete resorption was observed in four control grafts only. Cyst‐like resorption areas were more frequent within the graft of sham‐treated animals versus PEMF‐treated. The average volume of the cysts was not significantly different between the two groups; nevertheless, analysis of the variance of the volumes demonstrated a significant difference. The histological score showed no significant differences between controls and stimulated animals, but the percentage of surface covered by fibrous tissue was higher in the control group than in the stimulated one. Interleukin‐1 and tumor necrosis factor‐α concentration in the synovial fluid was significantly lower, and transforming growth factor‐β1 was significantly higher, in PEMF‐treated animals compared to controls. One month after osteochondral graft implantation, we observed larger bone formation in PEMF‐treated grafts which favors early graft stabilization. In the long term, PEMF exposure limited the bone resorption in subchondral bone; furthermore, the cytokine profile in the synovial fluid was indicative of a more favorable articular environment for the graft. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:631–642, 2008     

Periosteum‐derived mesenchymal progenitor cells in engineered implants promote fracture healing in a critical‐size defect rat model

An attractive alternative to bone autografts is the use of autologous mesenchymal progenitor cells (MSCs) in combination with biomaterials. We compared the therapeutic potential of different sources of mesenchymal stem cells in combination with biomaterials in a bone nonunion model. A critical‐size defect was created in Sprague–Dawley rats. Animals were divided into six groups, depending on the treatment to be applied: bone defect was left empty (CTL); treated with live bone allograft (LBA); hrBMP‐2 in collagen scaffold (CS^BMP2); acellular polycaprolactone scaffold (PCL group); PCL scaffold containing periosteum‐derived MSCs (PCL^PMSCs) and PCL containing bone marrow‐derived MSCs (PCL^BMSCs). To facilitate cell tracking, both MSCs and bone graft were isolated from green fluorescent protein (GFP)‐transgenic rats. CTL group did not show any signs of healing during the radiological follow‐up (n = 6). In the LBA group, all the animals showed bone bridging (n = 6) whereas in the CS^BMP2 group, four out of six animals demonstrated healing. In PCL and PCL^PMSCs groups, a reduced number of animals showed radiological healing, whereas no healing was detected in the PCL^BMSCs group. Using microcomputed tomography, the bone volume filling the defect was quantified, showing significant new bone formation in the LBA, CS^BMP2, and PCL^PMSCs groups when compared with the CTL group. At 10 weeks, GFP positive cells were detected only in the LBA group and restricted to the outer cortical bone in close contact with the periosteum. Tracking of cellular implants demonstrated significant survival of the PMSCs when compared with BMSCs. In conclusion, PMSCs improve bone regeneration being suitable for mimetic autograft design.     

自体全厚皮片内前列腺素E2含量变化对黑色素细胞密度的影响

目的：测定自体全厚皮片内前列腺素E2含量的动态变化，并观察其含量变化对黑色素细胞（MC）增殖的可能影响。方法：借助放射免疫分析法测定皮片PGE2含量，同时观察表皮内两种MC密度的动态变化。结果：术后7d以前，PGE2含量明显升高，而同期的MC密度经历了减少——缓慢增加这样一个损伤——修复过程。术后1月时，PGE2再次升高不伴MC密度的相应升高，但MC对^3H-TdR的摄取明显升高，表明其处于较强的复制状态。结论：我们有理由相信，PGE2含量的升高对于MC密度的恢复乃至超过术前水平所产生的作用是非常重要的。进一步研究PGE2的功能，并从控制皮片内的PGE2含量入手控制MC增殖，进而控制皮片的色泽，将是大有希望的途径之一。     

受区皮肤磨削后移植表皮治疗白癜风75例临床观察

目的 观察表皮细胞移植术治疗白癜风的疗效。方法 采用皮肤削进行自体有皮移值术。结果 治疗７５例白癜风患者共８５８区，治愈６８６区（７９．９５％），显效１０８区（１２．５９％），无效６４区（７．４６％），总有效率９２．５４％。结论 此法避免了液氮冷冻及斑蝥制剂等发疱造成的表皮不易成活等不利因素，且方便、灵活、治愈率高。     

Peripheral Blood Stem Cell Autografts in Children with Acute Lymphoblastic Leukemia and Lymphoma: Updated Experience

Peripheral blood stem cells (PBSC) in autografts (PBSCT) were given to 16 children with high-risk acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma (NHL). Harvest of PBSC is a safe, reliable procedure with low morbidity in children with cancer, and cryopreserved PBSC are useful in reducing cytopenia following marrow-ablative chemotherapy. The CFU-GM content of the thawed grafts is an important determinant of hematopoietic recovery after PBSCT. Whereas not all collections from children who had received intensive chemotherapy yielded sufficient progenitors for a safe graft, the value of preleukapheresis use of granulocyte colony-stimulating factor to expand the transplantable stem cell pool was not proven. After marrow-ablative chemotherapy without total body irradiation, three children who underwent PBSCT at refractory relapse died within 3 months. However, eight of the remaining 13 children are currently alive and disease-free, 5-29 months after PBSCT.     

Articular cartilage nutrition is mediated by subchondral bone: a long-term autograft study in baboons

OBJECTIVE: To determine the relative importance of subchondral nutrition in cartilage in autologous transplants and its relation to the development of osteoarthritis (OA). METHOD: The study was performed in non-human primates with two types of autografts placed orthotopically. One type of autograft was placed into vascularized, viable cancellous bone well, and another in an identical bone well, but coated with methylmethacrylate. The latter prevented direct contact between the autograft and the host bone. Observations were continued for 3 years. RESULTS: Abrogation of the contact between subchondral bone and articular cartilage-bone autograft had little effect on the cartilage during the first 5-12 months. By 3 years, autografts in the methylmethacrylate wells had non-vascularized and non-viable subchondral bone. The cartilage in these wells underwent degenerative changes compatible with OA. CONCLUSION: Interruption of contact between articular cartilage and vascularized subchondral bone resulted in degeneration of the cartilage. The onset and detection of these degenerative changes required long time periods (3 years). Had the experiments been terminated at 1 year or sooner the above described changes would not be apparent.