Successful fat-only whole breast reconstruction using cultured mature adipocytes and conditioned medium containing MCP-1.

A mastectomy is a curative treatment for breast cancer. It causes breast and soft tissue deficits, resulting in a chest with poor vascularity. Autologous tissue breast reconstruction is commonly associated with donor site morbidity. Breast implants are another reconstruction alternative, but they are associated with infection, rupture, and the need for replacement. Autologous aspirated fat grafting has appeared as an ideal breast reconstruction method, but low graft viability and high resorption remain as the main shortcomings. We developed a novel method for fat-only grafts using cultured mature adipocytes (CMAs) mixed with their condition medium. Twenty-five mastectomy patients, aged 32-72 years, received a mixed grafting of CMAs, MCP1-containing condition medium, and fat grafts for total breast reconstruction. In follow-up periods of 24-75 months, MRI analysis showed full thickness fat-engraftment. The cell proliferation marker Ki67 was negative in post-transplant biopsy specimens from all patients. Aesthetic full breast morphology was achieved, patient satisfaction was evaluated 1 year and 3-6 years after surgery. All grafts were confirmed safe, demonstrating high reliability and long-term sustainability.

Long-Term Clinical Results of Two-Stage Total Ear Reconstruction of Microtia Using Autologous Cell-Engineered Chondrocytes.

BACKGROUND: Microtia repair requires a large volume of reconstruction material.In pediatric patients, the collectable volume of autologous cartilage is limited, and the impact of surgical invasion and donor-site morbidity can be particularly severe. The authors developed a new treatment method using cultured autologous human auricular chondrocytes that provides a sufficiently large volume of reconstruction material. METHODS: Approximately 1 cm2 of auricular cartilage was collected from the affected site. Chondrocytes were isolated and cultured with autologous serum to accelerate cell proliferation. The cells were subcultured and formed a gel-form mass without a scaffold. In our two-stage implantation, the cultured chondrocytes were first injected into the patient's lower abdomen, where the cells grew into a large, newly generated cartilage in 6 months. Thereafter, this cartilage was sculpted into an ear framework and subcutaneously reimplanted into the new ear location. Clinical outcomes were assessed over a long-term follow-up. RESULTS: Eight patients underwent surgery using cultured autologous auricular chondrocytes from 2002 to 2008. The patients' ages ranged from 6 to 10 years. The follow-up period ranged from 11 to 18 years. None of the patients experienced absorption of cultured chondrocytes after the second stage. Complications included one case of absorption and one case of allergic reaction in the first stage. CONCLUSIONS: The authors' patients represent the first successful cases of regenerative surgery for microtia using cultured chondrocytes. No malignant transformation, change in size, deformation, or other abnormalities were observed during the long-term follow-up, demonstrating the safety of cultured cartilage. No major complications occurred. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Generative surgery of cultured autologous auricular chondrocytes for nasal augmentation.

BACKGROUND: Conventional treatment for nasal augmentation utilizes autologous grafts, allografts, or synthetic implants such as silicon implants. Silicon implants could protrude/expose or induce nasal bone resorption. Autologous grafts are usually associated with donor site morbidity and the volume of harvested tissue is limited. We had developed a new method for nasal augmentation using cultured autologous chondrocytes (CAC). The current report presents the results of a study using that method with a larger number of patients and an improved graft technique for the nasal tip. METHODS: Approximately 1 cm2 of cartilage was harvested from the auricular concha and treated with collagenase, and then chondrocytes were obtained. In our multilayer culture system the chondrocytes formed immature cartilaginous tissues with a gelatinous chondroid matrix. They were injection-grafted into the subcutaneous pocket of the nasal dorsum. RESULTS: The chondrocytes with a gelatinous chondroid matrix change from a soft gel to hard neocartilage tissue within 2 to 3 weeks and then stabilize. The authors have used this procedure over a 6-year period on 75 cases: 58 secondary augmentation rhinoplasties following silicon implantation and 17 primary augmentation cases. The results have been satisfactory and long-lasting. CONCLUSION: Grafting of CAC is an optional method for nasal augmentation and could be used for a wide range of facial augmentation cases.

Cryopreserved cultured epithelial allografts for pediatric deep partial dermal burns: Early wound closure and suppression of scarring.

BACKGROUND: In deep partial thickness dermal burns (DDB) where greater than 50% of the dermis is lost, severe pain, scarring and contractures occur. Therefore, skin grafting may be required. In children, scar contracture occurs because scarred skin does not stretch with growth creating the need for additional scar-releasing or skin-grafting surgeries. In order to resolve this problem, we used cryopreserved cultured epithelial allograft (cryopreserved allo-CEG), which can be grafted shortly after sustaining a wound. We reevaluated the promotion of early wound closure of burns and suppression of scarring by this treatment. METHODS: Cryopreserved allo-CEGs were used to treat 50 cases of pediatric DDB from 1992 to 2000. These cases were reviewed with regard to the time until epithelialization, take percentage, and pain level. Also, in order to examine why cryopreserved allo-CEG promotes healing of burns and suppresses scarring, growth factors and cytokines in the cryopreserved allo-CEG were measured. Cryopreserved allo-CEG sheets were solubilized and concentrations of TGF-α, TGF-ß1, IL-1α, IL-1ß, PDGF-AA, VEGF, KGF, IL-6, b-FGF, as well as metalloprotease-1 (MMP-1) and HGF, which are noted to have scarring suppression effects, were measured before grafting. RESULTS: Grafting of cryopreserved allo-CEGs in 50 cases of childhood DDB resulted in early epithelialization (9.32 ± 3.63 days on the average) and an almost 100% take rate. Also, pain relief (pain reduction or elimination, reduced need for anesthetics) was seen in all cases. Although 15-23 years have now elapsed, adverse events have not been observed. Cryopreserved allo-CEG contains IL-1α, IL-1ß, PDGF-AA, TGF-α, TGF-ß1, VEGF, and IL-6 have wound healing effects. The concentration of IL-1α was higher than the concentrations of other components, and this was followed by TGF-α, TGF-ß1, b-FGF and VEGF. Although the concentration of MMP-1, which has a scarring suppression effect, was high, HGF was not detected. CONCLUSION: Cryopreserved allo-CEG contains growth factors that promote wound healing and factors that suppress scarring. Three effects, namely (1) early wound closure, (2) scarring suppression, and (3) pain relief were seen with grafts of cryopreserved allo-CEG in cases of childhood DDB. These observations show that cryopreserved allo-CEG is clinically useful and effective for the treatment of childhood DDB.

[Anesthetic and perioperative management of patient for breast reconstruction in an outpatient clinic].

BACKGROUND: Our clinic provides various reconstructive surgeries but does not have facilities for patient admission, but there is a hospital providing satisfactory post-operative treatments in our area. METHODS: During the past 3 years, 276 patients received reconstructive mammoplasty under general anesthesia at our clinic. Their post-operative conditions immediately after the recovery were evaluated with the Modified Post-Anesthesia Discharge Scoring System (MPADSS). After observation for 4 to 5 hours in a recovery room, all the patients were transferred to another hospital by car. RESULTS: The score was 9 or 10 points in 45% of the patients. This indicates that the patients can be transferred to another hospital without problems. Twenty percent of patients had 6-7 points. These patients had mild bleeding, pain and/or unsteadiness/nausea/vomiting while walking. Among the 256 patients who were transferred to another hospital by car, one patient vomited in the car and 5 patients had nausea after the transfer. The other 20 patients whose score was 7 or lower were transferred by special vehicle with a stretcher and their conditions did not aggravate at all. CONCLUSIONS: At present, it is dangerous to perform all breast reconstructive surgeries as a day surgeries. A system is indispensable in which patients are transferred after surgery to another hospital with over-night stay facilities enabling observation.

Correction of secondary deformity after Nuss procedure for pectus excavatum by means of cultured autologous cartilage cell injection.

INTRODUCTION: For some cases of pectus excavatum, ideal chest shape cannot be achieved solely by performing the Nuss procedure. This manuscript presents a case where the residual deformity following Nuss was corrected using injection-transplantation of cultured autologous chondrocytes. PRESENTATION OF CASE: The treatment was performed for an 18-year-old male, who sought improvement of his chest shape after previously undergoing the Nuss procedure. A 1cm(2) auricular cartilage piece was harvested from his ear. Chondrocytes were isolated from the cartilage piece and were cultured. The cultured chondrocytes were processed into gel form and were injection-transplanted to the deformed region of the patient's chest. The grafted chondrocytes consolidated in one month, presenting elasticity equivalent to ordinary costal cartilage. The patient's chest remains in an optimal shape after a one-year postoperative follow up. DISCUSSION: Secondary correction of the chest deformity after previous operation for pectus excavatum is often tricky, because of the possible adhesion of the lungs or pericardium with the thoracic wall. Transplantation of cultured autologous chondrocytes does not require intra-thoracic maneuvers, and so is less invasive than other surgical interventions. Hence, priority can be placed, in some cases, on the chondrocyte transplantation rather than the re-correction of the thorax with the Nuss procedure or Ravitch procedure. CONCLUSION: Transplantation of cultured autologous chondrocytes is recommended as a useful option for secondary correction of chest deformity after the Nuss procedure.

Minimally invasive functional reconstruction after extended oropharyngeal resection including soft palate and base of tongue using a pectoralis major myocutaneous flap.

Excision of large oropharyngeal carcinomas that affect the base of the tongue and the soft palate severely impairs swallowing and articulation. In the present study we describe a minimally invasive technique that effectively restores swallowing and articulation by the insertion of a pectoralis major myocutaneous flap with a bilobular skin island. One lobe of the skin island is used to reconstruct the base of the tongue and the other to reconstruct the oropharynx. The soft palate is reconstructed by folding the tip of the lobe that is used to reconstruct the oropharynx in half along the long axis to fill the rhinopharynx. We have done this procedure for 13 patients with oropharyngeal carcinoma. Six months postoperatively all 13 were able to swallow without aspiration. Nine of the 13 patients were able to hold a normal conversation, but the remaining four had severe rhinolalia aperta. However, this condition was easily corrected by secondary reconstruction using a pharyngeal flap and a palatal mucoperiosteal flap (n = 3) or by the use of a small speech aid (n = 1).

Two-stage transplantation of cell-engineered autologous auricular chondrocytes to regenerate chondrofat composite tissue: clinical application in regenerative surgery.

BACKGROUND: The authors have developed a unique multilayered culture method that expands to large volumes elastic chondrocytes from a small piece of human auricular cartilage. In this study, the authors applied the two-stage transplantation method for cultured auricular chondrocytes to difficult cases of nasal/chin reconstruction where subcutaneous tissue is thin or scarred. METHODS: Auricular chondrocytes were cultured and expanded to sufficiently large volumes, and then, in a two-stage transplantation process, injection-transplanted into a patient's lower abdomen, where they were regenerated into larger chondrofat composite tissue in 6 months and used as a material for nasal/chin reconstruction. The authors then performed histologic and electron microscopic analysis of serial cross-sections and magnetic resonance imaging analysis of the chondrofat composite tissue. RESULTS: The cultured auricular chondrocytes consistency regenerated intraabdominally to a larger, stable neocartilage, with adherent fat tissue within 6 months. Eighteen patients (nose, n = 14; chin, n = 4) underwent this procedure, and the chondrofat composite tissue was stable after 1 to 5 years' postoperative follow-up. The chondrofat composite tissue maintained good shape, with no major complications. Magnetic resonance imaging showed that the chondrofat composite tissue was regenerated and vascularized in the abdomen in all 18 cases (100 percent). Infection and total absorption were not seen. Only partial absorption was noted (5.6 percent). CONCLUSIONS: The chondrofat composite tissue was found to be a new innovative graft material in which neocartilage is regenerated to be continuous with fat tissue by means of the neoperichondrium. It has thereby become possible to perform the previously impossible simultaneous reconstruction of cartilage and fat tissue.

Cell-engineered human elastic chondrocytes regenerate natural scaffold in vitro and neocartilage with neoperichondrium in the human body post-transplantation.

We have developed a unique method that allows us to culture large volumes of chondrocyte expansion from a small piece of human elastic cartilage. The characteristic features of our culturing method are that fibroblast growth factor-2 (FGF2), which promotes proliferation of elastic chondrocytes, is added to a culture medium, and that cell-engineering techniques are adopted in the multilayered culture system that we have developed. We have subsequently discovered that once multilayered chondrocytes are transplanted into a human body, differentiation induction that makes use of surrounding tissue occurs in situ, and a large cartilage block is obtained through cartinogenesis and matrix formation. We have named this method two-stage transplantation. We have clinically applied this transplantation method to the congenital ear defect, microtia, and reported successful ear reconstruction. In our present study, we demonstrated that when FGF2 was added to elastic chondrocytes, the cell count increased and the level of hyaluronic acid, which is a major extracellular matrix (ECM) component, increased. We also demonstrated that these biochemical changes are reflected in the morphology, with the elastic chondrocytes themselves producing a matrix and fibers in vitro to form a natural scaffold. We then demonstrated that inside the natural scaffold thus formed, the cells overlap, connect intercellularly to each other, and reconstruct a cartilage-like three-dimensional structure in vitro. We further demonstrated by immunohistochemical analysis and electron microscopic analysis that when the multilayered chondrocytes are subsequently transplanted into a living body (abdominal subcutaneous region) in the two-stage transplantation process, neocartilage and neoperichondrium of elastic cartilage origin are regenerated 6 months after transplantation. Further, evaluation by dynamic mechanical analysis showed the regenerated neocartilage to have the same viscoelasticity as normal auricular cartilage. Using our multilayered culture system supplemented with FGF2, elastic chondrocytes produce an ECM and also exhibit an intercellular network; therefore, they are able to maintain tissue integrity post-transplantation. These findings realized a clinical application for generative cartilage surgery.

Generating ears from cultured autologous auricular chondrocytes by using two-stage implantation in treatment of microtia.

BACKGROUND: Microtia is a congenital ear hypoplasia associated with auricular defects. Conventional treatment involves implanted costal cartilage. The impact of surgical invasion and donor-site morbidity can be particularly severe in pediatric patients, and the collectable volume of autologous cartilage is limited. The authors therefore developed a new technique for microtia and applied it to treat four patients. METHODS: Through the development of a multilayer chondrocyte culture system and two-stage implantation technique, the authors successfully generated human ears. In culture, the chondrocytes are expanded to a sufficiently large volume, produce rich chondroid matrix, and form immature cartilaginous tissues. In the authors' two-stage implantation, the cultured chondrocytes are injection-implanted into the lower abdomen of the patient, where the cells grow into a large, newly generated cartilage with neoperichondrium in 6 months. This cartilage is harvested surgically, sculptured into an ear framework, and implanted subcutaneously into the position of the new ear. RESULTS: The cultured chondrocytes formed a mature cartilage block with sufficient elasticity for use as an auricular cartilage. The formed block had the same histologic origin as elastic cartilage. The ear framework produced from this block was implanted into the auricular defect area, and an auricle with a smooth curvature and shape was subsequently configured. In the 2 to 5 years of postoperative monitoring, the neocartilage maintained good shape, without absorption. CONCLUSIONS: The authors' four patients are the first successful cases of regenerative surgery for microtia using cultured ear chondrocytes. The benefits of the technique include minimal surgical invasion, lower donor-site morbidity, lessened chance of immunologic rejection, and implantation stability.

Clinical application of cultured autologous human auricular chondrocytes with autologous serum for craniofacial or nasal augmentation and repair.

BACKGROUND: The repair of a craniofacial or nose deformity requires a large volume of reconstructive material. A conventional cartilage graft does not provide a sufficient volume of reconstructive material. Therefore, augmentation of the facial form to the defect shape is quite difficult. The authors developed a new treatment method that provides a sufficiently large volume of reconstructive material and enables an easier reconstruction of the original shape. METHODS: Ages of the patients ranged between 9 and 63 years. Approximately 1 cm of auricular cartilage was collected from the auricular concha. Isolated chondrocytes were cultured with autologous serum that accelerates cell proliferation. The cells were subcultured and formed a gel-form mass. This mass, together with autologous serum, was grafted (injected) on the periosteum and into the subcutaneous pocket. The volume of grafted cultured chondrocytes ranged from 1.7 to 40 cc (1 to 5 x 10(7) cells/cc). The lesion changed from soft gel form into hard cartilage tissues within 2 to 3 weeks and stabilized. RESULTS: Excellent or good satisfactory results were obtained in all patients and have been maintained for periods ranging from 3 to 34 months. No patient experienced absorption of cultured chondrocytes. Biopsy of the newly formed tissues showed that it was an elastic cartilage derived from the original tissue. CONCLUSIONS: A small number of chondrocytes obtained from a 1-cm auricular cartilage are successfully cultured into a large number of cells in a gel form. Those autologous auricular chondrocytes in a gel form allow for the repair of complicated shapes of the defect area. This technique is applicable to various treatments for craniofacial or nose deformity.

Fibrin stimulates the proliferation of human keratinocytes through the autocrine mechanism of transforming growth factor-alpha and epidermal growth factor receptor.

In the field of dermatology and plastic and reconstructive surgery, fibrin gel is regarded as a material that promotes wound healing. To test the hypothesis that fibrin may promote the growth of the epidermis, we examined its effects on the proliferation of cultured keratinocytes. Human keratinocytes were cultivated in fibrin-coated wells, and the cell numbers and transforming growth factor (TGF)-alpha, secreted into the cultured medium, were measured. We also assessed the capacity of epidermal growth factor receptor (EGF-R) that is responsible for all known actions of TGF-alpha and epidermal growth factor. The keratinocytes increased dramatically in their number, and the TGF-alpha secretion and the binding capacity of EGF-R were also increased dramatically in the presence of fibrin. These findings suggest that fibrin supports the proliferation of keratinocytes in an autocrine fashion via EGF-R; namely, fibrin stimulates keratinocytes to secrete TGF-alpha, which in turn increases cell proliferation and EGF-R capacity. We propose that fibrin can support the wound healing process of the epidermis via the TGF-alpha/EGF-R pathway.

Clinical application of biotechnically cultured autologous chondrocytes as novel graft material for nasal augmentation.

A new method of nasal augmentation has been developed, in which cultured autologous chondrocytes are transplanted. Using biotechnology, a piece of the choncha cartilage 1 cm2 is cultured into a gel-type mass of chondrocytes, which then is transplanted by injection into a surgically created subperiosteal skin pocket on the nasal dorsum. The augmented nose is taped and protected for 1 week. The grafted chondrocytes develop into mature cartilaginous tissue after approximately 1 month. This method was used in eight cases of nasal augmentation, and one case of chin augmentation (performed simultaneously), and one case of depressed deformity on the forehead. The results obtained by this method to date have been satisfactory after a follow-up time of 6 to 24 months. The authors believe that this method may at least partially be able to replace silicone implantation for nasal augmentation.