3D bioprinting of a gelatin-alginate hydrogel for tissue-engineered hair follicle regeneration.

Hair follicle (HF) regeneration remains challenging, principally due to the absence of a platform that can successfully generate the microenvironmental cues of hair neogenesis. Here, we demonstrate a 3D bioprinting technique based on a gelatin/alginate hydrogel (GAH) to construct a multilayer composite scaffold simulating the HF microenvironment in vivo. Fibroblasts (FBs), human umbilical vein endothelial cells (HUVECs), dermal papilla cells (DPCs), and epidermal cells (EPCs) were encapsulated in GAH (prepared from a mixture of gelatin and alginate) and respectively 3D-bioprinted into the different layers of a composite scaffold. The bioprinted scaffold with epidermis- and dermis-like structure was subsequently transplanted into full-thickness wounds in nude mice. The multilayer scaffold demonstrated suitable cytocompatibility and increased the proliferation ability of DPCs (1.2-fold; P < 0.05). It also facilitated the formation of self-aggregating DPC spheroids and restored DPC genes associated with hair induction (ALP, ß-catenin, and α-SMA). The dermal and epidermal cells self-assembled successfully into immature HFs in vitro. HFs were regenerated in the appropriate orientation in vivo, which can mainly be attributed to the hierarchical grid structure of the scaffold and the dot bioprinting of DPCs. Our 3D printed scaffolds provide a suitable microenvironment for DPCs to regenerate entire HFs and could make a significant contribution in the medical management of hair loss. This method may also have broader applications in skin tissue (and appendage) engineering. STATEMENT OF SIGNIFICANCE: Hair loss remains a challenging clinical problem that influences quality of life. Three-dimensional (3D) bioprinting has become a useful tool for the fabrication of tissue constructs for transplantation and other biomedical applications. In this study, we used a 3D bioprinting technique based on a gelatin/alginate hydrogel to construct a multi-layer composite scaffold with cuticular and corium layers to simulate the microenvironment of dermal papilla cells (DPCs) in the human body. This new approach permits the controllable formation of self-aggregating spheroids of DPCs in a physiologically relevant extracellular matrix and the initiation of epidermal-mesenchymal interactions, which results in HF formation in vivo. The ability to regenerate entire HFs should have a significant impact on the medical management of hair loss.

Microenvironmental reprogramming of human dermal papilla cells for hair follicle tissue engineering.

The restoration of hair-inductive potential in human dermal papilla cells (hDPCs) is a tremendous challenge for hair regeneration. Much of the research thus far has indicated that three-dimensional (3-D) culture shows improved efficacy in hair follicle (HF) neogenesis. However, mature HF cannot regenerate in an incomplete microenvironment. This study developed an optimized 3-D co-culture system to restore the hair-inductive characteristics of hDPCs by mimicking the in-vivo microenvironment. As a result, Matrigel-encapsulated hDPCs spontaneously formed into hDPC aggregates (hDPAs), which exhibited better activity, higher proliferation rates, and less apoptosis and hypoxia than the ultra-low attachment culture. Interestingly, the co-culture with the hair matrix cells and dermal sheath cup cells further enhanced the expression of hair regeneration-related genes of hDPAs compared to conditioned medium and improved mature HF induction. In addition, these hDPAs with higher hair inductivity could be produced on a large scale and easily separated for gene expression detection. Finally, the mRNA sequencing, PCR, and WB results showed that the co-culture biomimetic microenvironment stimulated the canonical Wnt signaling pathway and inhibited the BMP signaling pathway. Thus, this co-culture system will provide a reliable platform that allows high-throughput culture, testing, and harvesting of hDPAs for HF tissue engineering. STATEMENT OF SIGNIFICANCE: Extensive hair loss continues to be difficult to treat and causes significant patient morbidity. Hair follicle (HF) tissue engineering may seem to be a way out. However, the absence of the in-vivo microenvironment fails to regenerate mature hairs. This study systematically described a biomimetic co-culture approach to generate better quality human dermal papilla cell aggregates (hDPAs) with improved hair inductive properties, which can be further used for HF tissue engineering. The hDPC microenvironment was reprogrammed through the controllable formation of self-assembled organoids in Matrigel and the tri-culture with hair matrix cells and dermal sheath cup cells. This work indicates that the production of hDPAs could be readily scaled, in theory for large-scale assays, analyses, or therapeutic applications.

A three-dimensional bioprinting technique, based on a gelatin/alginate hydrogel, for the tissue engineering of hair follicle reconstruction.

Hair loss remains a challenging clinical problem that influences the quality of life. Three-dimensional (3D) bioprinting has become a valuable tool for fabricating tissue constructs for transplantation and other biomedical applications. Although some simple organs, such as skin and cartilage, have been successfully simulated, it remains challenging to make hair follicles (HFs), which are highly complex organs. The tissue engineering of human HFs has been a long-standing challenge, and progress with this has lagged behind that with other lab-grown tissues. This is principally due to a lack of availability of a platform that can successfully recapitulate the microenvironmental cues required to maintain the requisite cellular interactions for hair neogenesis. In this study, we used a 3D bioprinting technique based on a gelatin/alginate hydrogel to construct a multilayer composite scaffold with cuticular and corium layers to simulate the microenvironment of dermal papilla cells (DPCs) in the human body. This new approach permits the controllable formation of self-aggregating spheroids of DPCs in a physiologically relevant extracellular matrix and the initiation of epidermal-mesenchymal interactions, which results in HF formation in vivo. In conclusion, our 3D-bioprinted multilayer composite scaffold prepared using a gelatin/alginate hydrogel provides a suitable 3D microenvironment for DPCs to induce HF formation. The ability to regenerate entire HFs should have a significant impact on the medical management of hair loss. This method may also have critical applications for skin tissue engineering, with its appendages, for other purposes.

Validity and reliability of three-dimensional costal cartilage imaging for donor-site assessment and clinical application in microtia reconstruction patients: A prospective study of 22 cases.

OBJECTIVES: This study assesses the ability to reconstruct costal cartilage images by using three-dimensional visualisation software (Mimics) based on semi-automated segmentation algorithm and to investigate its reliability and validity with an anthropometric analysis. DESIGN: Observational prospective study. SETTING: Plastic surgery department of a tertiary hospital. PARTICIPANTS: Twenty-two microtia patients who underwent autologous ear reconstruction. MAIN OUTCOME MEASURES: Preoperative thoracic computed tomography data were processed to Mimics software for three-dimensional costal cartilage imaging. The length, width, thickness and volume of the 9th costal cartilages were calculated from these images and compared with the direct measurements (DM) obtained intraoperatively. RESULTS: The intra-examiner reliability and inter-examiner reliability were high in terms of all four measurements (intraclass correlation coefficients, ICC: 0.876-0.984). There were no significant differences between image-based anthropometry and DM in the linear measurements except for the volume (P < .05). The mean volume calculation error of Mimics was -0.08 ± 0.13 mL. No correlation was found between the anthropometric variables and the absolute errors (P > .05). Furthermore, Bland-Altman plots were used to evaluate the agreement between the two methods. CONCLUSIONS: Despite a very small error was found in volume calculation, Mimics software was accurate and reliable in linear calculation. Three-dimensional costal cartilage imaging was found to be an efficient tool for morphological evaluation of costal cartilages. We believe that with the application of individualised cartilage models based on three-dimensional printing, the use of customised ear framework carving will be practicable in surgical training.

Which Fat Processing Can Achieve Optimal Transplantation in Patients With Insufficient Fat Resource?

BACKGROUND: Autologous fat transfer has been widely used in breast augment surgery. The breast fat graft is different form facial, it needs megavolume grafts. However, most Asian women are thin, who often encounter the fat resource insufficiency during breast augmentation. In fact, some processing methods do contribute to great loss of potential fat that could have been grafted. How to choose a best processing method to achieve optimal transfer in patients with insufficient fat resources is fairly important. METHODS: Lipoaspirate was obtained from 10 healthy female patients who underwent liposuction from abdomen. According to the processing methods, 10-mL initial fat grafts divided into 4 groups: decantation (group A), centrifugation (group B), cotton pad (group C), and cell-assisted lipotransfer (CAL) (group D). Lipoaspirate from each group was compared in the vitro and vivo experiments. The content and function of stromal vascular fraction (SVF) were compared as well as lipoaspirate survival after grafting in nude mice. RESULTS: The SVFs were counted in decantation group 4.32 ± 0.75 × 10/mL, centrifuge group 3.48 ± 0.78 × 10/mL, cotton pad group 1.64 ± 0.84 × 10/mL, CAL group 4.08 ± 0.73 × 10/mL. The decantation group was higher than the cotton pad group (P < 0.05). All the groups' SVFs had capability of multilineage differentiation. The fat graft weight in decantation group: 0.3908 ± 0.023 g, centrifuge group 0.3073 ± 0.015 g, cotton pad group 0.1726 ± 0.019 g, and CAL group 0.2396 ± 0.021 g. The weight of the fat graft in the decantation group was greater than that of the centrifugation group, cotton pad group and CAL group (P < 0.05). There was no significant difference in cell integrity, necrotic cysts and fibrosis between the groups. The vascularization degree in the cotton pad group was lower than that in the decantation group (P < 0.05). CONCLUSIONS: Decantation processing can achieve optimal transplantation in patients who have insufficient fat resources.

Locally Increased Level of Inorganic Phosphate Induced Nodules or Calcification After Bolus Fat Grafting.

BACKGROUND: Nodules or calcifications have been a common complication after breast augmentation with fat grafting, especially in cases with partial bolus fat grafting. There are some clinical preventive measures, but mechanisms related to this complication have not been elucidated yet. Inorganic phosphate (PI), being a product of fat metabolism, is a well-known stimulus of other kinds of pathological calcification such as vascular calcification. We aimed to determine whether PI had a similar effect on formation of nodules after fat grafting. METHODS: Nodules or calcification after fat grafting models using nude mice were created by bolus fat injection. Levels of PI of necrotic liquid located in the central zone and mineralization deposition of graft were examined 1 week, 2 weeks, 1 month, 2 months and 7 months after bolus fat injection. External high phosphate solution was injected 3 times a week to the fat grafts for 2 months, and mineral deposition was examined. In addition, adipose-derived stem cells (ADSCs) were treated with high phosphate osteogenic differentiation medium in various concentrations and times. ADSCs were also treated with osteogenic differentiation in addition to tetramisole which could reduce the level of PI. Mineral depositions of the cells were examined. The central necrotic liquid was extracted from patients who found palpable nodules after breast augmentation with fat grafting. The level of PI of this necrotic liquid and normal lipoaspirates from patients who received normal liposuction for body contouring was compared. RESULTS: The in vivo study indicated that the local PI concentration of the necrotic zone increased significantly 2 months after large volume bolus fat injection. Calcification was not formed after 2 months, but was formed after 7 months, indicating that the effect of PI on calcification was time-dependent. In addition, with the effect of external injection of high phosphate solution into the fat graft, calcification was formed after 2 months, indicating the effect of PI on calcification was dose-dependent. The in vitro study also indicated PI could induce calcification of ADSC in a time- and dose-dependent manner. The study in humans indicated that the level of PI in the necrotic zone of nodules after fat grafting was higher than that in normal lipoaspirates. CONCLUSIONS: This study indicated that the level of PI in the central necrotic zone was elevated after bolus fat injection, which could provide an environment to induce calcification of surrounding tissue. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

The Effect of Respiration on Breast Measurement Using Three-dimensional Breast Imaging.

BACKGROUND: Three-dimensional (3D) imaging offers new opportunities to enable objective and quantitative analysis of the breast. Unlike scanning of rigid objects, respiration may be one of the factors that can influence the measurement of breast when using 3D imaging. In this study, we aimed to investigate how the different respiratory phases affect 3D morphologic and volumetric evaluations of the breast. METHODS: We performed preoperative 3D breast imaging at the end of expiration (EE) and the end of inspiration (EI). We repeated scans on each respiratory phase, taking four scans in total (EE1, EE2 and EI1, EI2). Using Geomagic Studio 12 software, measurements from the different respiratory phases (EE1 and EI1) were compared for differences in the linear distances of breast. Breast volumetric change error (BVCE) was measured between EE1 and EE2 (R1) and between EI1 and EI2 (R2). A multilevel model was used to analyze the difference of linear-distances parameters between EE1 and EI1 and a paired sample t-test was used to analyze the difference between R1 and R2. RESULTS: Our study included 13 Chinese women (26 breasts) with a mean age of 32.6 ± 6.3 years. Compared with EI, EE showed a longer sternal notch to the level of the inframammary fold and shorter nipple to midline (p < 0.05). During EI, breast projection increased by 0.23 cm (95% CI - 0.39, - 0.08) and breast base width increased by 0.27 cm (95% CI - 0.46, - 0.09). The position of the nipple moved by 0.18 cm (95% CI - 0.34, - 0.03) laterally, 0.41 cm (95% CI 0.18, 0.64) cranially, and 0.71 cm (95% CI - 0.92, - 0.51) anteriorly. Although there was no significant difference in BVCE between EE and EI, the result seen with EE appeared to be more consistent. CONCLUSIONS: The results of this study demonstrate that there was no difference in breast volume results when patients are in the expiratory or inspiratory state during 3D breast imaging. This study, however, holds potential benefits to both surgical practice as well as the 3D imaging industry. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Fat Necrosis After Autologous Fat Transfer (AFT) to Breast: Comparison of Low-Speed Centrifugation with Sedimentation.

BACKGROUND: Despite the advantages of autologous fat transfer to the breast, there are many complications after the surgery, such as oil cysts, calcification and palpable breast nodules. The fat purification process is a key step to reduce those complications, but there is currently no standard processing method. This study was designed to compare the incidence of fat necrosis after autologous fat grafting to the breast with low-speed centrifugation and sedimentation. METHODS: This study analyzed 100 patients (167 breasts) who underwent autologous fat grafts to the breasts from January, 2015 to March, 2017. Patients were divided into two groups randomly, low-speed centrifugation (800 r/min) and sedimentation (15 min). Postoperative fat necrosis such as oil cysts and palpable breast nodules was measured using physical examination and breast ultrasound 3 months after the surgery. The number and the diameter of the fat necroses were detected. RESULTS: A total of 100 patients (167 breasts) were included this research. There were 21 breasts with clinically palpable nodules (12.57%); fifteen (19.48%) were in the low-speed centrifugation group and six (6.67%) were in the sedimentation group (p < 0.05). According to postoperative breast ultrasounds, there were 83 breasts with hypoechoic cyst formations (49.7%); forty-five (58.44%) in the low-speed centrifugation group and 38 (42.22%) in the sedimentation group (p < 0.05). A positive correlation between the number of operative sessions and fat necrosis was found out. CONCLUSION: Although low-speed centrifugation could achieve higher fat purification efficacy when compared with sedimentation, it causes more fat necrosis than sedimentation, such as clinically palpable nodules and subclinical nodules, especially in patients who had a history of breast surgery and those who needed more than one fat grafting session. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .