微囊化新生猪甲状旁腺细胞异种移植的实验研究

目的 探讨微囊化新生猪甲状旁腺细胞异种移植治疗大鼠甲状旁腺功能低下症的效果。方法 应用微囊化技术，制备微囊化（海藻酸钠－聚赖氨酸－海藻酸钠生物微胶囊）新生猪甲状旁腺细胞，32只去甲状旁腺的Wistar大鼠随机分成微囊组、非微囊组、空囊组和对照组，分别移植微囊化新生猪甲状旁腺细胞、甲状旁腺细胞、空微囊及生理盐水。移植后监测血钙及甲状旁腺素水平40周，40周后回收移植物，透射电镜检查。结果 移植后，微囊组大鼠血钙及甲状旁腺素水平恢复到正常范围内，直至观察结束时（40周），透射电镜检查显示移植物存活良好；非微囊组、空囊组和对照组大鼠的血钙及甲状旁腺素水平无改善。结论 微囊化新生猪甲状旁腺细胞异种移植在不用免疫抑制剂情况下，可以在大鼠体内存活，且有功能；海藻酸钠－聚赖氨酸－海藻酸钠生物微胶囊对免疫活性细胞及抗体具有屏蔽作用。     

Preoperative evaluation of microencapsulated human parathyroid tissue aids selection of the optimal bioartificial graft for human parathyroid allotransplantation

Allotransplantation of microencapsulated parathyroid tissue is a promising approach to the treatment of permanent hypoparathyroidism. Preoperative assessment of the quality of microencapsulated parathyroid tissue could facilitate selection of the optimal bioartifical graft for human parathyroid allotransplantation. Parathyroid tissue from patients with secondary hyperparathyroidism (n = 15) was processed mechanically or enzymatically (collagenase type II). Tissue particles and single cells/cell clusters were routinely microencapsulated with amitogenic Ba(2+) alginate. Parathyroid secretion dynamics in response to stimulation of nonencapsulated and microencapsulated parathyroid tissue with Ca(2+) were evaluated in a perifusion system. The stability of the different types of microcapsule was assessed using an osmotic pressure test. Mechanical cutting of parathyroid tissue led to peripheral necrosis of tissue particles and impaired their vitality. Collagenase digestion, in contrast, resulted in single cells and cell clusters without peripheral necrosis. The quality of microencapsulation of single cells/cell clusters was significantly better than that of tissue particles (deformed and imperfect capsules). Microencapsulation itself did not decrease cell vitality. Nonencapsulated and microencapsulated tissue particles and single cells/cell clusters from different donors maintained their own levels of response to stimulation with low Ca(2+). Microcapsules containing tissue particles showed poor stability compared with those containing single cells/cell clusters. Preoperative evaluation of microencapsulated parathyroid tissue can disclose differences in vitality and function and thus facilitate selection of the optimal bioartifical graft for human parathyroid allotransplantation.     

In Vitro and In Vivo Evaluation of Protamine–Heparin Membrane for Microencapsulation of Rat Langerhans Islets

Abstract: Rat pancreatic islets were microencapsulated with multilayer protamine–heparin (PH) membrane. Basal and stimulatory insulin secretion of microencapsulated islets was similar to the controlled free islets in vitro. During the long–term culture (up to 2 weeks) mean insulin release of encapsulated islets did not significantly differ from the mean of free ones (the ratio of mentioned means was 54–167%). Empty PH microcapsules transplanted into Wistar rats intraperitoneally and under the kidney capsule were generally harmless up to 4 months. In only a few cases traces of fibrotic tissue around capsules entrapped in the omentum were found. No damage of microcapsules structure was observed. The worst results were obtained in the instance of retroperitoneal transplantation. We conclude, therefore, that PH membrane was proved to be highly biocompatible, nontoxic for islets, and did not impair viability and glucose–dependent insulin secretion of Langerhans islets in in vitro culture.     

Survival of microencapsulated islets at 400 days posttransplantation in the omental pouch of NOD mice

The long-term durability of agarose microencapsulated islets against autoimmunity was evaluated in NOD mice. Islets were isolated from 6-8-week-old prediabetic male NOD mice and microencapsulated in 5% agarose hydrogel. Microencapsulated or nonencapsulated islets were transplanted into the omental pouch of spontaneously diabetic NOD mice. Although the diabetic NOD mice that received nonencapsulated islets experienced a temporary reversal of their hyperglycemic condition, all 10 of these mice returned to hyperglycemia within 3 weeks. In contrast, 9 of 10 mice transplanted with microencapsulated islets maintained normoglycemia for more than 100 days. Islet grafts were removed at 100, 150, 200, 300, and 400 days posttransplantation. A prompt return to hyperglycemia was observed in the mice after graft removal, indicating that the encapsulated islet grafts were responsible for maintaining euglycemia. Histological examination revealed viable islets in the capsules at all time points of graft removal. In addition, beta-cells within the capsules remained well granulated as revealed by the immunohistochemical detection of insulin. No immune cells were detected inside the microcapsules and no morphological irregularities of the microcapsules were observed at any time point, suggesting that the microcapsules successfully protected the islets from cellular immunity. Sufficient vascularization was evident close to the microcapsules. Considerable numbers of islets showed central necrosis at 400 days posttransplantation, although the necrotic islets made up only a small percentage of the islet grafts. Islets with central necrosis also showed abundant insulin production throughout the entire islets, except for the necrotic part. These results demonstrate the long-term durability of agarose microcapsules against autoimmunity in a syngeneic islet transplantation model in NOD mice.     

Parathyroid Xenotransplantation without Immunosuppression in Experimental Hypoparathyroidism: Long-term In Vivo Function following Microencapsulation with a Clinically Suitable Alginate

Permanent hypoparthyroidism is one of the most difficult of all endocrine disorders to treat medically. Because this deficiency syndrome rarely is a life-threatening condition, systemic immunosuppression for recipients of transgenic transplants is not justified. An alternative would be protecting the tissue to be transplanted from the immunologic response (immunoisolation) by coating it with a semipermeable membrane- microencapsulation. Unfortunately, prior to the first clinical use, further analysis of the coating substance (alginate) demonstrated that it has potential cancerogenic properties. Using a purified amitogenic alginate suitable for clinical use, we accomplished allotransplantation in a long-term animal model and reported the first clinical cases without postoperative immunosuppression recently. In view of the potential clinical use, we investigated the ability of the microencapsulation technology with the novel amitogenic alginate in experimental hypoparathyroidism (80 parathyroidectomized rats) to enable transgenic transplantation across the highest immunologic barrier (xenotransplantation: human to rat) to ensure intact transplant function without immunosuppression. In a controlled, long-term animal study, the effect of microencapsulation on xenotransplanted human parathyroid tissue was evaluated over a period of 30 weeks (microencapsulated and naked hyperplastic parathyroid tissue, respectively). Functionally, human parathyroid tissue was able to replace that of rats. More than 6 months after xenotransplantation 32 of 40 animals that had received microencapsulated transplants were normocalcemic. In contrast, serum calcium concentrations dropped to postparathyroidectomy levels within 3 weeks in the animals that had received naked tissue only. Correspondingly, normocalcemic animals showed vital parathyroid tissue inside the microcapsules, which were surrounded by a small rim of fibroblasts. When combining microencapsulation with an improved tissue culture method, xenotransplantation of human parathyroid tissue and maintenance of its physiologic function is reproducibly achieved over the highest transplantation barrier. Using the amitogenic alginate may be a crucial step toward the first clinical use of this technique for parathyroid xenotransplantation without immunosuppression.     

Long-term graft function of adult rat and human islets encapsulated in novel alginate-based microcapsules after transplantation in immunocompetent diabetic mice

We describe the results of the first study to show that adult rat and human islets can be protected against xenogenic rejection in immunocompetent diabetic mice by encapsulating them in a novel alginate-based microcapsule system with no additional permselective membrane. Nonencapsulated islets lost function within 4-8 days after being transplanted into diabetic Balb/c mice, whereas transplanted encapsulated adult rat or human islets resulted in normoglycemia for >7 months. When rat islet grafts were removed 10 and 36 weeks after transplantation, the mice became immediately hyperglycemic, thus demonstrating the efficacy of the encapsulated islets. The explanted capsules showed only a mild cellular reaction on their surface and a viability of >85%, and responded to a glucose stimulus with a 10-fold increase in insulin secretion. Furthermore, transplanted mice showed a slight decrease in the glucose clearance rate in response to intraperitoneal glucose tolerance tests 3-16 weeks after transplantation; after 16 weeks, the rate remained stable. Similar results were obtained for encapsulated human islets. Thus we provide the first evidence of successful transplantation of microencapsulated human islets. In conclusion, we have developed a novel microcapsule system that enables survival and function of adult rat and human islets in immunocompetent mice without immunosuppression for >7 months.     

Immune mechanisms associated with the rejection of encapsulated neonatal porcine islet xenografts

BACKGROUND: The immune mechanisms associated with the rejection of microencapsulated neonatal porcine islets (NPI) are not clearly understood. Therefore, in this study we characterized the immune cells and molecules that are involved in this process by examining the microencapsulated NPI xenografts at various time points post-transplantation in B6 mice. METHODS: Microencapsulated NPI were transplanted into streptozotocin-induced diabetic immune-competent B6 and immune-deficient B6 rag-/- mice and blood glucose levels were monitored twice a week. Encapsulated NPI were then recovered from B6 mice at various time points post-transplantation to characterize the islets and immune response using immunohistochemical and RT-PCR analyses. To determine which T-cell subpopulation is important for the rejection of encapsulated NPI, B6 rag-/- mice with established microencapsulated NPI xenografts were reconstituted with either CD4(+) or CD8(+) T cells and a return to the diabetic state was noted. For controls, adoptive transfer experiments involved reconstitution of B6 rag-/- mice with established microencapsulated NPI with non-fractionated lymph node cells or non-reconstituted mice. RESULTS: All B6 recipients of microencapsulated NPI remained diabetic throughout the study while B6 rag-/- recipients achieved normoglycemia and maintained normoglycemia for up to 100 days post-transplantation. Encapsulated NPI recovered from B6 mice at early time points (day 7 and day 14) post-transplantation were surrounded with very few layers of immune cells that increased with time post-transplantation. The extent of cellular overgrowth on the surface of encapsulated NPI has a significant correlation with islet cell death and the presence of CD4(+) T cells, B cells and macrophages. Mouse IgG antibody and complement as well as cytokines [gamma-interferon (IFN-gamma), interleukin10 (IL10)] and chemokines (monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha and beta) were detected within the microcapsules at several time points post-transplantation suggesting that these molecules can traverse the microcapsule. Mouse anti-porcine IgG antibodies in recipient sera were found to peak at 30 days post-transplantation indicating leakage of porcine xenoantigens. In contrast, microencapsulated NPI recovered from B6 rag-/- mice had no cellular overgrowth on the surface. Complement and cytokines (IL 10 but not IFN-gamma) including chemokines were detected within the microcapsules at several days post-transplantation. We also found that B6 rag-/- mice reconstituted with non-fractionated lymph node cells or CD4(+) T cells but not CD8(+) T cells became diabetic demonstrating that CD4(+) T cells are the necessary T-cell subtype for microencapsulated NPI rejection. In contrast, non-reconstituted B6 rag-/- mice remained normoglycemic for the entire duration of the study. CONCLUSIONS: Our results demonstrate that CD4(+) T cells, B cells and macrophages are the immune cells recruited to and involved in the rejection of encapsulated NPI. Immune molecules secreted by these cells as well as complement can traverse the microcapsule membrane and are responsible for destroying the NPI cells. Treatment regimens which target these molecules may modify the rejection of encapsulated NPI and lead to prolonged islet xenograft survival.     

Agarose enhances the viability of intraperitoneally implanted microencapsulated L929 fibroblasts

To achieve immunoisolation, mouse L929 fibroblasts were encapsulated in approximately 400 microm poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA) microcapsules and were subsequently implanted in the peritoneal cavity of syngeneic C3H mice. As a baseline for the use of genetically engineered cells in cell encapsulation therapy, the L929 cells were transfected to express a secreted form of human alkaline phosphatase (SEAP). Implantation of empty microcapsules in a PBS suspension resulted in deformation, aggregation, and poor retrievability of the microcapsules. Incubation of microcapsules with medium containing xenogeneic horse serum prior to implantation increased the thickness of the fibrous tissue surrounding the microcapsules. However, immobilization of the microcapsules in a 4% (w/v) SeaPlaque agarose gel prior to implantation allowed complete recovery of the microcapsules and prevented their aggregation and deformation. As a result, approximately 50% of the encapsulated cells remained viable 21 days postimplantation. Moreover, once the viable cells were released from retrieved microcapsules and regrown as monolayers, they expressed SEAP at a level similar to their encapsulated but nonimplanted counterparts.     

Prolonged glucose normalization of streptozotocin-induced diabetic mice by transplantation of rat islets coencapsulated with crosslinked hemoglobin

BACKGROUND: Facilitated oxygen transport by crosslinked hemoglobin (Hb-C) in islet microcapsules may promote transplanted graft function by improving islet functionality and viability. METHODS: This study investigated the in vivo efficacy of Hb-C as an oxygen carrier on the functionality and viability of microencapsulated rat islets. Hb-C by poly(ethylene glycol) was introduced into rat islet microcapsules (alginate-poly[L-lysine]-alginate microcapsule), and 500 suboptimal encapsulated islets were xenotransplanted into each streptozotocin-induced diabetic BALB/c mouse. The graft efficacy over time was evaluated by measuring nonfasting blood glucose level, body weight, and glucose tolerance. RESULTS: Mice that received Hb-C-containing microcapsules maintained normoglycemia for at least 8 weeks with normal glucose clearance, determined by intraperitoneal glucose tolerance test. However, the mice that received the conventional control islet microcapsule (without Hb-C) transplant showed graft failure in 4 weeks, exhibited by hyperglycemia, weight loss, and deteriorated glucose tolerance. Severe central necrosis of retrieved islets was observed for the control islet capsule graft after 8 weeks. CONCLUSION: The present study revealed that the incorporation of Hb-C in islet microcapsules promotes graft function for a longer period of time than the conventional islet capsules. Therefore, Hb-C coencapsulation is a potential approach for prolonging graft function of islet microcapsules and reducing the number of islets required for normoglycemia.     

Evidence for an antigen-release induced cellular immune response against alginate-polylysine encapsulated islets

Abstract: For almost 15 years microencapsulated islets have been successfully transplanted in different animal models. Besides long-lasting success, a certain rate of graft failures also has been reported. This study evaluated the cellular immune response toward alginate-polylysine microencapsulated islets (mc-islets) in vitro by means of mixed-lymphocyte-islet-culture. Method: Islets were isolated from LEWIS-rats and lymphocytes from the spleens of NMRl-mice. After overnight culture and prior to encapsulation the islets were gamma-irradiated with 22.5 Gy. 50 mc-islets, 50 nonencapsulated islets, and 50 empty me were co-cultured for 72 h with 1 × 10⁶ in 1 ml medium in 24-well plates. Thymidine incorporation during the last 24 hr served as a measure for lymphoid activation. Result: Non-encapsulated islets were potent stimulators of lymphoid proliferation. This could be reduced by 55% when the islets were entrapped in microcapsules. Empty microcapsules induced a minor lymphoid activation (18% of mc-islets) only. Conclusion: Despite encapsulation islets could be recognised by lymphocytes in vitro. This may explain at least partially graft failure rates after transplantation of encapsulated islets.     

Endothelin-1 regulates parathyroid hormone expression of human parathyroid cells

OBJECTIVE: Parathyroid cells synthesize and release endothelin-1 (ET-1). ET-1 displays an in vitro inhibitory effect on basal parathyroid hormone (PTH) secretion and also counteracts PTH hypersecretion stimulated by low calcium. Such effects are further demonstrated in vivo, independent of the changes in calcitonin. We propose that ET-1 may regulate the pathogenesis of uremic hyperparathyroidism. However, this was not directly demonstrated in human parathyroid glands. DESIGN: Hyperplastic parathyroid glands are obtained from the surgical operation for uremic hyperparathyroidism. Cells are isolated by enzyme digestion and treated with ET-1, and are assessed for PTH mRNA expression. PTH in the plasma and the medium is measured by a newly developed method to detect the whole PTH (1-84). PATIENTS: Uremic patients with severe secondary hyperparathyroidism and ultrasonography-proved hypertrophy of parathyroid glands received elective surgical approaches under general anesthesia. The resected glands were immediately taken to the laboratory for fresh isolation. MEASUREMENTS: Following ET-1 treatment, PTH mRNA expression is evaluated by RT-PCR method. ET-1 is detected with radioimmunoassay kit and PTH is measured by a new commercially available Duo PTH kit. RESULTS: ET-1 exhibited a dose-dependent inhibitory effect (from 10(-12) - 10(-7) M) on PTH mRNA expression of parathyroid cells, either in the basal or in the low-calcium-stimulated states. Release of PTH into the medium is also gradually inhibited by the increase in ET-1 concentrations. CONCLUSIONS: Our results demonstrate that ET-1 attenuates PTH mRNA expression in freshly isolated human parathyroid cells, and PTH release is also decreased. This result is consistent with our previously reported in vitro and in vivo experiments.     

微囊豚鼠肝细胞培养及其细胞免疫屏障作用的观察

目的 观察海藻酸钠-多聚赖氨酸-海藻酸钠(APA)微囊化肝细胞的生物学功能及对免疫细胞的隔离效应。方法 用胶原酶门腔静脉灌注法分离豚鼠肝细胞,测定微囊化肝细胞及游离肝细胞培养上清白蛋白水平,用自然杀伤细胞(NK)细胞的细胞毒实验来评价微囊的免疫隔离效应。结果 微囊包裹对肝细胞的活率无明显影响,微囊化肝细胞与裸露肝细胞白蛋白分泌水平差异无显著性(P>0.05),NK细胞对K562靶细胞的细胞毒实验表明微囊可有效地保护囊内细胞不受NK细胞的杀伤作用。结论 APA微囊化肝细胞可保持良好的生物活性,APA微囊对细胞免疫具有免疫隔离作用。     

海藻酸-壳聚糖-聚乙烯乙二醇微囊生物相容性的研究

目的比较海藻酸-壳聚糖-聚乙烯乙二醇微囊(ACP微囊)和海藻酸-聚赖氨酸-海藻酸微囊(APA微囊)的生物相容性。方法(1)两种微囊(50、100和200个)与健康人血清共浴,检测微囊对补体的激活程度。(2)1000个APA和ACP微囊分别植入Wistar大鼠的腹腔,4d和3周时统计取出的微囊数和微囊的纤维化率。(3)Wistar大鼠胰岛用ACP微囊和APA微囊包裹,分别贯续置于含3.3mmol/L和16.7mmol/L葡萄糖的Hank's溶液中培养,测定培养液中胰岛素的浓度。结果(1)ACP微囊组残余补体活性高于APA微囊组。(2)4d时,ACP和APA微囊的取出数分别是845.0±40.4和807.6±45.7(P>0.05),囊周纤维化率分别是16.40%和65.68%(P<0.05);3周时两种微囊的取出数分别为715.0±133.0和367.5±105.6(P<0.05),囊周纤维化率为27.8%和83.9%(P<0.05)。(3)在含3.3mmol/L葡萄糖的Hank's液中,未微囊胰岛组、APA和ACP微囊化胰岛组的胰岛素浓度分别是(123.48±4.70)mIU/L、(110.11±12.18)mIU/L和(110.90±11.95)mIU/L,当葡萄糖浓度为16.7mmol/L时,胰岛素浓度分别是(754.75±13.81)mIU/L、(689.30±27.71)mIU/L和(684.28±70.10)mIU/L。结论海藻酸-壳聚糖-聚乙烯乙二醇微囊的生物相容性要优于海藻酸-聚赖氨酸-海藻酸微囊,前者更适合应用于微囊化胰岛移植。     

The alginate-poly-L-lysine-alginate membrane: Evidence of a protective effect on microencapsulated islets of Langerhans following exposure to cytokines

Abstract: In past experiments, we demonstrated that allografts and xenografts of pancreatic islets encapsulated in alginate-polylysine-alginate (APA) microcapsules were protected from immunorejection and that in both streptozotocin-induced and spontaneously diabetic animals diabetes was reversed for extended periods of time. Because of growing evidence that cytokines may play an important role in graft rejection, it is important to determine whether the APA capsular membrane can also provide a protective barrier against cytotoxic actions of cytokines. In this study, free and encapsulated rat islets were exposed in vitro to interleukin 1, 2, and 6 (IL-1, IL-2, and IL-6) to tumor necrosis factor (TNF) and interferon (INT), then challenged with 20 mM glucose for 24 hr to evaluate physiological response in terms of insulin secretion. Although IL-2 and 6 were found to have no effect on the pancreatic islets (free or encapsulated), IL-1, TNF, and INT were shown to interfere with the physiological pattern of insulin secretion from the free islets; microencapsulated islets' function was not affected. Transmission electron microscopy (TEM) of the rat islets exposed to the five types of cytokines revealed that IL-1, TNF, and INF exerted morphological changes to the free islets while encapsulated islets were not affected. No changes were detected in free or encapsulated islets exposed to IL-6 or IL-2. In addition, encapsulated islets were transplanted into normal BALB-c mice and into spontaneously diabetic NOD mice for 3 to 5 months and then recovered. The recovered islets were shown to continue to respond to glucose challenge in a physiological manner. The results of this study showed that, although exposure of some cytokines to free islets results in changes in the normal physiological response of the islets to glucose challenge as well as in ultrastructural changes within the cells, microencapsulated islets were not impaired by the exposure to the same cytokines.     

Experimental microencapsulation of porcine and rat pancreatic islet cells with air-driven droplet generator and alginate

Transplantation of microencapsulated islets is proposed as an ideal therapy for the treatment of type 1 diabetes mellitus without immunosuppression. This strategy is based on the principle that foreign cells are protected from the host immune system by an artificial membrane. The aim of this study was to establish an ideal condition of microencapsulation using an air-driven droplet generator and alginate in vitro. The optimal conditions for islet encapsulation were an alginate inflow rate of 10 mL/h, CO₂ flow rate of 2.0 L/min in a concentration of 2% alginate. For 2.5% alginate, the alginate inflow rate of 20 mL/h, CO₂ flow rate 3.0 L/min was ideal; alginate inflow rate of 40 mL/h, CO₂ flow rate of 4.0 L/min showed good microcapsules at 3% alginate. Viability of encapsulated islets was greater than 90%. In terms of insulin secretion, encapsulated islets secreted insulin in response to glucose in static culture medium. However, there was no normal response to low or high glucose challenge with a stimulation index less than 2.0. Microencapsulation of pig islets was successfully performed with air-driven droplet generator and alginate in vitro. Further studies about biocompatibility and glucose control in vivo may provide a useful tool for treatment of patients with diabetes mellitus.     

Xenotransplantation of Microencapsulated Canine Islets into Diabetic Rats

Abstract: Islets of Langerhans were isolated in high yields from canine pancreata. In the procedure, the pan-creata were perfused and digested with collagenase, and the islets were then purified on histopaque density gradients. As many as 60,000 islets were isolated from a single pancreas. Islets were encapsulated in alginate-polylysinealginate membranes with the aid of an air-jet droplet generator. In vitro studies demonstrated that the isolated and encapsulated islets secreted insulin in response to glucose and IBMX challenge for at least 9 weeks. In in vivo studies 6 diabetic Wistar rats were transplanted with 5,000 to 8,000 encapsulated islets each. The diabetic condition was reversed in all recipients for up to 112 days. In control animals, which received free, unencapsulated islets, the xenografts remained functional for fewer than 21 days. Microcapsules retrieved from normoglycemic transplant recipients 1 and 2 months posttransplantation were shown to contain viable islet tissue, and no cellular overgrowth was observed on capsular surfaces. The results of the study indicate a considerable clinical potential of microencapsulated canine islet xenografts.     

Differentiation of transplanted microencapsulated fetal pancreatic cells

BACKGROUND: Fetal beta cells are a potential form of cell therapy for type 1 diabetes. To protect transplanted cells from cellular immune attack, microencapsulation using barium alginate can be employed. Whether microencapsulated fetal pancreatic cells will differentiate as occurs with nonencapsulated fetal pancreatic cells is presently unknown. It is suggested that such differentiation would occur in encapsulated cells, similar to previous experiments conducted using encapsulated embryonic stem cells. METHODS: Streptozotocin-induced diabetic severe combined immunodeficient mice were transplanted with 5,000 to 38,000 fetal pig islet-like cell clusters (ICCs) within barium alginate microcapsules of diameter 300, 600, or 1000 microm. Viability, insulin secretion, and content of encapsulated cells were measured prior to transplantation. Blood glucose levels (BGL) were measured twice weekly and porcine C-peptide monthly. Encapsulated cells were recovered from mice at 6 months posttransplantation for analysis. RESULTS: Encapsulated cells became glucose responsive and normalized BGL within 13 to 68 days posttransplantation, with 5,000 to 10,000 ICCs required. Microcapsule diameter did not affect the time required to achieve normoglycemia. BGL remained normal for the 6-month duration of the experiments. After removal of grafts at 25 weeks posttransplantation, glucose stimulated insulin secretion of the explants was enhanced 96-fold, insulin content was enhanced 34-fold, and the percentage of insulin and glucagon positive cells increased 10-fold and threefold, respectively, from the time of transplantation. CONCLUSIONS: This study demonstrates that fetal pancreatic cells differentiate and function normally when placed within barium alginate microcapsules and transplanted.     

微囊化人头皮毛乳头细胞移植诱导毛发形成

目的探索微囊化人头皮毛乳头细胞（以下简称毛乳头细胞）对裸鼠背部皮肤毛囊形成的诱导作用。方法胶原酶消化法体外分离、培养毛乳头细胞，再将毛乳头细胞以海藻酸钠-多聚赖氨酸-海藻酸钠（alginate-polylysine—alginate，APA）微囊包裹，以胶原凝胶作为载体，植入裸鼠背部皮下；移植空囊、游离毛乳头细胞各作为对照。观察移植部位毛发生长情况，利用组织学方法观测所形成的毛囊结构。结果微囊化毛乳头细胞皮下移植4周后，裸鼠背部移植区有白色、浓密、分布均匀的毛发长出。局部组织切片见大量发育完整的毛囊结构；而空囊及游离毛乳头细胞移植均未能诱导出上述现象。结论聚集性生长的微囊化毛乳头细胞能够保持诱导皮肤毛囊形成的作用，且这种作用无种属特异性。     

A novel, noninvasive imaging technique for intraoperative assessment of parathyroid glands: confocal reflectance microscopy

BACKGROUND: Successful surgical management of primary hyperparathyroidism requires the ability to identify and distinguish normal from abnormal parathyroid tissue. Microscopic pathologic confirmation often helps with the diagnoses and decisions regarding the extent of parathyroid resection. Confocal reflectance microscopy (CRM) is an optical method of noninvasively imaging tissue without fixation, sectioning, and staining as in standard histopathology. The goal of this study was to determine if CRM imaging could be used to distinguish normal from diseased parathyroid tissue intraoperatively. METHODS: In this study, 44 parathyroid glands from 21 patients undergoing operations for primary hyperparathyroidism were imaged immediately after excision. CRM images were compared with conventional hematoxylin-and-eosin stained sections obtained from the same gland. The percentage area occupied by fat cells was calculated in images of both normal and diseased glands. RESULTS: Characteristic microscopic features of parathyroid glands were distinguishable by CRM and correlated well with histopathology. The stromal fat content of normal and diseased glands could easily be determined. The percentage area occupied by fat cells differed significantly (P <.00001) in normal glands (average, 23.0% +/- 10.9%) and adenomatous glands (average, 0.4% +/- 0.7%). CONCLUSIONS: CRM imaging rapidly revealed microscopic features that reliably differentiated normal and diseased parathyroid glands. The success of this preliminary ex vivo study promotes interest in further development of an in situ probe for in vivo clinical diagnostic use.     

In vitro suppression of parathyroid hormone secretion by 22-oxa-calcitriol in human parathyroid hyperplasia due to uraermia

SUMMARY: 22-oxa-calcitriol (OCT), a vitamin D analogue, suppresses parathyroid hormone (PTH) secretion and has less calcaemic activity than 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] in vivo. In this study, we evaluated the effect of OCT on PTH secretion in vitro using human hyperplastic parathyroid tissue obtained during surgery for advanced renal hyperparathyroidism and normal bovine parathyroid glands to compare the effects of 1,25(OH)₂D₃. 22-oxa-calcitriol suppressed PTH secretion by nodular hyperplastic parathyroid tissue and normal bovine tissue in a dose dependent manner, the same as 1,25(OH)₂D₃. We showed the additive effect of extracellular calcium level on suppression of PTH secretion by OCT and 1,25(OH)₂D₃. These results suggest that OCT suppresses PTH secretion, the same as 1,25(OH)₂D₃ not only in normal parathyroid cells but also on hyperplastic parathyroid cells.