Establishment of Three‐Dimensional Tissue‐engineered Bone Constructs Under Microgravity‐simulated Conditions

Bone constructs have been grown in vitro with use of isolated cells, biodegradable polymer scaffolds, and bioreactors. In our work, the relationships between the composition and mechanical properties of engineered bone constructs were studied by culturing bone marrow mesenchymal stem cells (BMSCs) on ceramic bovine bone scaffolds in different environments: static flasks and dynamic culture system in rotating vessels—which was a National Aeronautics and Space Administration‐recommended, ground‐based, microgravity‐simulating system. After 15 days of cultivation, osteogenicity was determined according to DNA and alkaline phosphatase (ALP) analysis. DNA content and ALP were higher for cells grown on dynamic culture. Subsequently, the two kinds of engineered bone constructs were selected for transplantation into Sprague‐Dawley rat cranial bone defects. After 24 weeks of in vivo implantation, the engineered bone constructs under dynamic culture were found to repair the defects better, with the engineered constructs showing histologically better bone connection. Thus, this dynamic system provides a useful in vitro model to construct the functional role and effects of osteogenesis in the proliferation, differentiation, and maturation of BMSCs. These findings suggest that the hydrodynamic microgravity conditions in tissue‐culture bioreactors can modulate the composition, morphology, and function of the engineered bone.     

Cardiovascular tissue engineering I. Perfusion bioreactors: a review

Tissue engineering is a fast-evolving field of biomedical science and technology with future promise to manufacture living tissues and organs for replacement, repair, and regeneration of diseased organs. Owing to the specific role of hemodynamics in the development, maintenance, and functioning of the cardiovascular system, bioreactors are a fundamental of cardiovascular tissue engineering. The development of perfusion bioreactor technology for cardiovascular tissue engineering is a direct sequence of previous historic successes in extracorporeal circulation techniques. Bioreactors provide a fluidic environment for tissue engineered tissue and organs, and guarantee their viability, maturation, biomonitoring, testing, storage, and transportation. There are different types of bioreactors and they vary greatly in their size, complexity, and functional capabilities. Although progress in design and functional properties of perfusion bioreactors for tissue engineered blood vessels, heart valves, and myocardial patches is obvious, there are some challenges and insufficiently addressed issues, and room for bioreactor design improvement and performance optimization. These challenges include creating a triple perfusion bioreactor for vascularized tubular tissue engineered cardiac construct; designing and manufacturing fluidics-based perfused minibioreactors; incorporation of systematic mathematical modeling and computer simulation based on computational fluid dynamics into the bioreactor designing process; and development of automatic systems of hydrodynamic regime control. Designing and engineering of built-in noninvasive biomonitoring systems is another important challenge. The optimal and most efficient perfusion and conditioning regime, which accelerates tissue maturation of tissue-engineered constructs also remains to be determined. This is a first article in a series of reviews on critical elements of cardiovascular tissue engineering technology describing the current status, unsolved problems, and challenges of bioreactor technology in cardiovascular tissue engineering and outlining future trends and developments.     

Tissue engineering of osteochondral constructs in vitro using bioreactors

Articular cartilage is a relatively simple tissue, but has a limited capacity of restoration. Tissue engineering is a promising field that seeks to accomplish the in vitro generation of complex, functional, 3-dimensional tissues. Various cell types and scaffolds have been tested for these purposes. The results of tissue engineered cartilage and bone are as yet inferior to native tissue. Strain and perfusion have been shown to stimulate cell proliferation and differentiation of various cell phenotypes. The perfect protocol to produce articular cartilage has not been defined yet. Bioreactors could provide the environment to engineer osteochondral constructs in vitro and to provide a stress protocol. The bioreactor has to provide an economically viable approach to automated manufacture of functional grafts under clinical aspects. Composite engineered tissues, like an engineered joint, represent a future goal. Cross-disciplinary approaches are necessary in order to succeed in engineering osteochondral grafts that provide adequate primary biomechanical stability and incorporate rapidly in vivo with histological appearance close to healthy osteochondral tissue. This review surveys current clinical and experimental concepts and discusses challenges and future expectations in this advancing field of regenerative medicine focusing human osteochondral constructs in bioreactors.     

Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.

Cartilaginous constructs have been grown in vitro with use of isolated cells, biodegradable polymer scaffolds, and bioreactors. In the present work, the relationships between the composition and mechanical properties of engineered cartilage constructs were studied by culturing bovine calf articular chondrocytes on fibrous polyglycolic acid scaffolds (5 mm in diameter, 2-mm thick, and 97% porous) in three different environments: static flasks, mixed flasks, and rotating vessels. After 6 weeks of cultivation, the composition, morphology, and mechanical function of the constructs in radially confined static and dynamic compression all depended on the conditions of in vitro cultivation. Static culture yielded small and fragile constructs, while turbulent flow in mixed flasks yielded constructs with fibrous outer capsules; both environments resulted in constructs with poor mechanical properties. The constructs that were cultured freely suspended in a dynamic laminar flow field in rotating vessels were the largest, contained continuous cartilage-like extracellular matrices with the highest fractions of glycosaminoglycan and collagen, and had the best mechanical properties. The equilibrium modulus, hydraulic permeability, dynamic stiffness, and streaming potential correlated with the wet-weight fractions of glycosaminoglycan, collagen, and water. These findings suggest that the hydrodynamic conditions in tissue-culture bioreactors can modulate the composition, morphology, mechanical properties, and electromechanical function of engineered cartilage.     

Bioreactors for tissue engineering--a new role for perfusionists?

Tissue engineering is an exciting new area of medicine with rapid growth and expansion over the last decade. It has the potential to have a profound impact on the practice of medicine and influence the economic development in the industry of biotechnology. In almost every specialty of medicine, the ability to generate replacement cells and develop tissues will change the focus from artificial organs and transplantation to growing replacement organs from the patient's own stem cells. Once these organs are at a size that requires perfusion to maintain oxygen and nutrient delivery, then automated perfusion systems termed "bioreactors" will be necessary to sustain the organ until harvesting. The design of these "bioreactors" will have a crucial role in the maintenance of cellular function throughout the growth period. The perfusion schemes necessary to determine the optimal conditions have not been well elucidated and will undergo extensive research over the next decade. The key to progress in this endeavor will development of long-term perfusion techniques and identifying the ideal pressures, flow rates, type of flow (pulsatile/nonpulsatile), and perfusate solution. Perfusionists are considered experts in the field of whole body perfusion, and it is possible that they can participate in the development and operation of these "bioreactors." Additional education of perfusionists in the area of tissue engineering is necessary in order for them to become integral parts of this exciting new area of medicine.     

Recovery of preservation-injured primary human hepatocytes and nonparenchymal cells to tissuelike structures in large-scale bioreactors for liver support: an initial transmission electron microscopy study.

This study investigated large-scale regeneration and tissue reorganization of adult human liver cells from preservation injured transplant organs. The use of basement membrane protein gels and growth factor enriched culture medium in standard culture flasks promotes liver tissue formation in isolated rat and pig hepatocytes, resulting in prolongation of phenotypic stability and metabolic competence of primary cells in vitro. A special bioreactor construction for high-density three-dimensional cell recovery was developed and isolation of cells from discarded human donor livers was enabled. In vitro regeneration of adult human liver cells isolated from preservation-injured organs took place over a period of 2 weeks in a purpose-built bioreactor. Basement membrane protein and growth factors were avoided. Reorganization of tissue structures was studied using transmission electron microscopy (TEM). This showed regeneration and tissue reorganization of adult human cells from preservation-injured organs by coculture with nonparenchymal cells in the bioreactor. The majority of the aggregated hepatocytes in the bioreactors showed morphological similarities to those in vivo (although not re-formed to hepatocyte plates), exhibiting cell-cell junctions and reconstituted bile canaliculi-like spaces between neighboring hepatocytes. Perfusion channels appeared regularly between cell aggregates. The arrangement of nonparenchymal cells between the hepatocyte aggregates exhibited similarities to liver sinusoids. Endothelial cells often covered the aggregates and formed a borderline to the perfusion channels between the capillaries. Similar to the space of Disse, further nonparenchymal cells were located between the endothelial cells and the parenchymal aggregates. Deposits of biomatrix fibers occurred spontaneously. The regenerated cell mass was close to that of a single liver lobe. In conclusion, the further optimization of bioreactors that enable cell recovery from preservation injury may lead to the utilization of cells from discarded whole or split transplants for extracorporeal temporary liver support therapy or hepatocyte transplantation.     

利用灌注式生物反应器体外构建大段组织工程化骨的实验研究

目的 研究体外利用灌注式生物反应器构建大段组织工程化骨的可行性. 方法把在体外培养扩增的第三代人骨髓基质干细胞与大段多孔β-磷酸三钙(β-TCP)支架复合.将细胞/支架复合体放入灌注式生物反应器中,进行连续灌注培养.28 d后,检测细胞的增殖及碱性磷酸酶(ALP)活性,同时对培养后的细胞/支架复合体进行组织学检测及形态学计量,用以评价体外组织工程化骨的构建.以静态培养作为对照组. 结果培养28 d后,灌注培养组的细胞活性明显高于静态培养组.灌注培养组细胞的ALP活性显著高于静态培养组.静态培养组细胞仅在多孔β-TCP支架周缘增殖,形成的新骨量较少.灌注培养组细胞在整个β-TCP支架内增殖,形成的新骨量较多. 结论利用灌注式生物反应器的灌注培养,可以使人骨髓基质干细胞在大段β-TCP载体内增殖并形成新骨,使体外大段组织工程化骨的构建成为可能.     

A perfusion bioreactor for intestinal tissue engineering

BACKGROUND: Short gut syndrome is a devastating clinical problem with limited long-term treatment options. A unique characteristic of the normal intestinal epithelium is its capacity for regeneration and adaptation. Despite this tremendous capacity in vivo, one of the major limitations in advancing the understanding of intestinal epithelial differentiation and proliferation has been the difficulty in maintaining primary cultures of normal gut epithelium in vitro. A perfusion bioreactor system has been shown to be beneficial in long-term culture and bioengineering of a variety of tissues. The purpose of this study is to design and fabricate a perfusion bioreactor for intestinal tissue engineering. MATERIALS AND METHODS: A perfusion bioreactor is fabricated using specific parameters. Intestinal epithelial organoid units harvested from neonatal rats are seeded onto biodegradable polymer scaffolds and cultured for 2 d in the bioreactor. Cell attachment, viability, and survival are assessed using MTT assay, scanning electron micrograph, and histology. RESULTS: A functional perfusion bioreactor was successfully designed and manufactured. MTT assay and scanning electron micrograph demonstrated successful attachment of viable cells onto the polymer scaffolds. Histology confirmed the survival of intestinal epithelial cells seeded on the scaffolds and cultured in the perfusion bioreactor for 2 days. CONCLUSIONS: A functional perfusion bioreactor can be successfully fabricated for the in-vitro cultivation of intestinal epithelial cells. With further optimization, the perfusion bioreactor may be a useful in in-vitro system for engineering new intestinal tissue.     

The effects of simulated microgravity on intervertebral disc degeneration

Background contextAstronauts experience back pain, particularly low back pain, during and after spaceflight. Recent studies have described histologic and biochemical changes in rat intervertebral discs after space travel, but there is still no in vitro model to investigate the effects of microgravity on disc metabolism.PurposeTo study the effects of microgravity on disc degeneration and establish an in vitro simulated microgravity study model.Study designDiscs were cultured in static and rotating conditions in bioreactor, and the characteristics of disc degeneration were evaluated.MethodsThe mice discs were cultured in a rotating wall vessel bioreactor where the microgravity condition was simulated. Intervertebral discs were cultured in static and microgravity condition. Histology, biochemistry, and immunohistochemical assays were performed to evaluate the characteristics of the discs in microgravity condition.ResultsIntervertebral discs cultured in rotating bioreactors were found to develop changes of disc degeneration manifested by reduced red Safranin-O staining within the annulus fibrosus, downregulated glycosaminoglycan (GAG) content and GAG/hydroxyproline ratio, increased matrix metalloproteinase 3 expression, and upregulated apoptosis.ConclusionsWe conclude that simulated microgravity induces the molecular changes of disc degeneration. The rotating bioreactor model will provide a foundation to investigate the effects of microgravity on disc metabolism.     

A new bioartificial liver using porcine hepatocyte spheroids in high-cell-density suspension perfusion culture: in vitro performance in synthesized culture medium and in 100% human plasma

A prototype of a bioartificial liver (BAL) based on suspension perfusion culture of porcine hepatocyte spheroids was developed at 150 ml scale. About 2% (4 x 10(9) cells) of whole human liver cells was immobilized. The cell density in the bioreactor was 2.7 x 10(7) cells/ml, which was almost comparable to that of presently developed packed-bed-type BALs. The bioreactor was perfused with culture medium while retaining spheroids. This was done using a rotating stainless filter (pore size 50 microm). In vitro 8-h perfusion experiments utilizing both synthesized culture medium and 100% human plasma demonstrated the spheroids in the bioreactor had almost the same functions on a unit/cell basis as those in small-scale rotational culture. This indicated that the functional deterioration often associated with scaling up had been minimized. Rapid spheroid aggregation and dysfunction in specific human plasma pool must be eliminated before clinical application, although this phenomenon seemed to be inherent to porcine hepatocyte-based BALs. This prototype shows promise in meeting present clinical demands by achieving maximal metabolic activities even in the short term.     

旋转生物反应器内微载体扩增骨髓间充质干细胞组织工程法修复兔下颌骨缺损

目的探索旋转生物反应器内微载体扩增骨髓间充质干细胞(BMSCs)组织工程法及BM-SCs与PHB的复合支架修复兔下颌骨缺损的可行性。方法体外分离纯化BMSCs,在旋转生物反应器内,利用微载体将其在短时间内快速扩增后接种于聚羟基丁酸酯(PHB)支架上,以修复兔的下颌骨缺损区,此为实验A组;以未对缺损区进行修复为对照B组;以单纯PHB修复缺损区为对照C组。分别于修复术后第3、8、14、42、84天处死每组兔2只,对缺损区行组织学检查、骨形态发生蛋白(BMP)免疫组织化学检查、X线摄片。结果第84天,A组的大部分修复材料被骨性组织取代;B组的缺损区未修复;A组修复骨缺损的效率较C组高。结论在旋转生物反应器内,应用微载体技术可以成功地进行BMSCs的培养和快速扩增,能满足骨组织工程的需要;PHB可以作为一种组织工程材料修复骨缺损。     

Does mechanical stimulation have any role in urinary bladder tissue engineering?

INTRODUCTION: Tissue engineering of the urinary bladder currently relies on biocompatible scaffolds that deliver biological and physical functionality with negligible risks of immunogenic or tumorigenic potential. Recent research suggests that autologous cells that are propagated in culture and seeded on scaffolds prior to implantation improve clinical outcomes. For example, normal urinary bladder development in utero requires regular filling and emptying, and current research suggests that bladders constructed in vitro may also benefit from regular mechanical stimulation. Such stimulation appears to induce favorable cellular changes, proliferation, and production of structurally suitable extracellular matrix (ECM) components essential for the normal function of hollow dynamic organs. MATERIALS AND METHODS: To mimic in vivo urinary bladder dynamics, tissue bioreactors that imitate the filling and emptying of a normal bladder have been devised. A "urinary bladder tissue bioreactor" that is able to recapitulate these dynamics while providing a cellular environment that facilitates cell-cell and cell-matrix interactions normally seen in-vivo may be necessary to successfully engineer bladder tissue. CONCLUSIONS: The validation of a urinary bladder tissue bioreactor that permits careful control of physiological conditions will generate a broad interest from researchers interested in urinary bladder physiology and tissue engineering.     

Rotating three‐dimensional dynamic culture of adult human bone marrow‐derived cells for tissue engineering of hyaline cartilage

The method of constructing cartilage tissue from bone marrow‐derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow‐derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per µg DNA of the tissues were 10.01 ± 3.49 µg/µg DNA in the case of the RWV bioreactor and 6.27 ± 3.41 µg/µg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three‐dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow‐derived cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 517–521, 2009     

Semipermeable Hollow Fiber Membranes in Hepatocyte Bioreactors: A Prerequisite for a Successful Bioartificial Liver?

Abstract: Recent studies have shown that liver support systems based on viable hepatocytes can prolong life in animal models of acute liver failure. Now the time has come to elucidate the design characteristics that are essential to construct an efficient bioreactor. The gold standard remains the intact liver. Despite the very high cell density in this organ, individual cell perfusion is guaranteed resulting in low diffusional gradients which are essential for optimal mass transfer. These conditions are not met in bioreactors based on hollow fiber membranes. Moreover, the semipermeable membranes can foul and act as a diffusional barrier between the hepatocytes and the blood or plasma of the recipient. We devised a novel bioreactor for use as a bioartificial liver that does not include hollow fiber membranes for blood or plasma perfusion. The device is based on an integral oxygenator and a nonwoven polyester matrix material for hepatocyte culture as small aggregates. The efficacy of this original design was tested in rats with liver ischemia. Preliminary results show statistically significantly improved survival; life was prolonged 100% compared to the control experiments.     

Bioreactor microcarrier cell culture system (Bio-MCCS) for large-scale production of autologous melanocytes

Restoration of cutaneous pigmentation can be achieved in stable vitiligo by autologous cultured melanocyte transplantation. It was the goal of this study to construct a bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous melanocytes in large scale. In this Bio-MCCS, porcine gelatin microbeads were used as microcarriers, spinning bottle as fermented tank. Autologous melanocytes were able to attach to and proliferate on the gelatin microbeads in serum-free melanocyte medium in the Bio-MCCS, reaching up to 24-fold the cells seeded on day 15 (MTT assay). These autologous melanocytes cultured on gelatin microbeads could leave the microbeads and proliferate on the bottom of tissue culture flasks. Although Pluronic F68 has been widely used to protect animal cells from hydrodynamic stress in animal cell bioreactors, Pluronic F68 at a concentration of 0.25-1.0% showed no significant protective effects on the autologous melanocytes cultured on the microbeads and subjected to mechanical stress in the Bio-MCCS. This Bio-MCCS using porcine gelatin microbeads as microcarriers enabled large-scale production of autologous melanocytes, offering a potential treatment for large-area stable vitiligo by direct administration of the melanocytes cultured on the gelatin microbeads to the vitiliginous site.     

Relevance of bioreactors and whole tissue cultures for the translation of new therapies to humans

The purpose of this review is to provide a brief overview of bioreactor‐based culture systems as alternatives to conventional two‐ and three‐dimensional counterparts. The role, challenges, and future aspirations of bioreactors in the musculoskeletal field (e.g., cartilage, intervertebral disc, tendon, and bone) are discussed. Bioreactors, by recapitulating physiological processes, can be used effectively as part of the initial in vitro screening, reducing that way the number of animal required for preclinical assessment, complying with the 3R principles and, in most cases, allowing working with human tissues. The clinical significance of bioreactors is that, by providing more physiologically relevant conditions to customarily used two‐ and three‐dimensional cultures, they hold the potential to provide a testing platform that is more predictable of a whole tissue response, thereby facilitating the screening of treatments before the initiation of clinical trials. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:10–21, 2018.     

Adult human articular chondrocytes in a microcarrier‐based culture system: expansion and redifferentiation

Expanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use microcarriers as substrates for chondrocyte expansion. While microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer‐expanded cells. Cells left attached to microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:539–546, 2011     

A brief history of tendon and ligament bioreactors: Impact and future prospects

Bioreactors are powerful tools with the potential to model tissue development and disease in vitro. For nearly four decades, bioreactors have been used to create tendon and ligament tissue-engineered constructs in order to define basic mechanisms of cell function, extracellular matrix deposition, tissue organization, injury, and tissue remodeling. This review provides a historical perspective of tendon and ligament bioreactors and their contributions to this advancing field. First, we demonstrate the need for bioreactors to improve understanding of tendon and ligament function and dysfunction. Next, we detail the history and evolution of bioreactor development and design from simple stretching of explants to fabrication and stimulation of two- and three-dimensional constructs. Then, we demonstrate how research using tendon and ligament bioreactors has led to pivotal basic science and tissue-engineering discoveries. Finally, we provide guidance for new basic, applied, and clinical research utilizing these valuable systems, recognizing that fundamental knowledge of cell-cell and cell-matrix interactions combined with appropriate mechanical and chemical stimulation of constructs could ultimately lead to functional tendon and ligament repairs in the coming decades.     

组织工程软骨实验生物反应器的设计

目的:设计一套计算机控制的组织工程软骨实验用灌流型生物反应器。方法:通过计算机控制时间及流量,利用蠕动泵、密闭的硅胶管、玻璃管、储液瓶等串联设计一套无菌的灌流型软骨组织工程用生物反应器。结果:生物反应器由控制系统和培养系统组成,运行良好,能置于培养箱中对软骨细胞材料复合物进行动态培养。结论:反应器设计合理。整个生物反应器系统运行可靠。     

Wound healing on athymic mice with engineered skin substitutes fabricated with keratinocytes harvested from an automated bioreactor

The Kerator is a computer controlled bioreactor for the automated culture and harvest of keratinocytes that can reduce labor and materials involved in the fabrication of engineered skin substitutes (ESS). Previous studies have shown that the Kerator is comparable to tissue culture flasks by keratinocyte confluence during culture, clonogenic potential of harvested keratinocytes and microanatomy, cell viability, and surface hydration of ESS fabricated with the harvested keratinocytes. In this study, the Kerator and tissue culture flasks were further compared by keratinocyte proliferation in vitro and wound healing after transplantation of ESS to athymic mice. The number of bromodeoxyuridine-positive keratinocytes in ESS fabricated with keratinocytes harvested from Kerator after 2 wk of in vitro maturation was 34 +/- 3 per high power field (hpf) (mean +/- SEM), which was not significantly different from that fabricated with keratinocytes harvested from flasks (34 +/- 1.5 per hpf). Percentage original wound area 6 wk after surgery of ESS fabricated with keratinocytes from the Kerator was 36% +/- 3.3%, which was not significantly different from that of ESS fabricated with keratinocytes from flasks (30% +/- 4.3%). In both cases, 78% (7 of 9) mice transplanted were positive for engraftment of human keratinocytes by direct immunofluorescence for HLA-ABC antigens. These results further confirm that the ESS fabricated with keratinocytes harvested from Kerator and flasks are equivalent in vitro and in vivo. Therefore, use of Kerator for large scale production of ESS can lead to increased availability at reduced cost while maintaining ESS quality for grafting.