Cell Modification in 3D: a new Paradigm in the Creation of Living Cell-Biomaterial Composites

我们的目标是开发新的、改进的材料和方法，使干细胞（间充质细胞；MSCs）能够被操纵形成骨组织。我们将使用微小的（<100纳米；人类头发直径的1/1000）磷酸钙（羟基磷灰石；HAP）颗粒作为载体，将特定的生物分子携带到细胞中。为了最大限度地传递这些化学和遗传信号，每个细胞的整个细胞表面将被载体覆盖（3D涂层）。具体来说，这将使我们能够生产和生长自我支持的活骨组织，无论是在体内受损部位，还是在体外准备植入的培养皿中。更一般地说，这种方法的效率和成本效益的提高也将促进从间质干细胞生成其他组织类型（例如神经和肌肉）以及修饰其他类型干细胞的研究。为什么是间充质干细胞？干细胞作为治疗剂具有巨大的潜力。胚胎干细胞（ESC）具有形成体内所有主要类型细胞的潜力，并且在培养中相对容易生长。然而，有道德和兼容性问题的使用。成体干细胞（ASC）可以从特定的组织类型（血液、神经、皮肤）中获得，但需要扩大群体以获得足够的材料用于治疗。在这方面，骨髓间充质干细胞（MSC）的分离和快速培养方法已经建立，为组织工程提供了很大的希望。它们是骨、软骨、脂肪和纤维结缔组织形成的天然前体。因此，它们已经作为替代受损骨组织（例如修复手术）系统的组成部分进行了深入研究。此外，同一个人可以是供体和受体，从而减轻了与胚胎干细胞有关的问题。为什么是小羟基磷灰石颗粒？羟基磷灰石是在骨骼中发现的磷酸钙的化学形式，因此它与细胞兼容。骨中的HAP晶体大小也相似（<100 nm）。此外，由于晶体非常小，并且覆盖在细胞上，一些晶体会在细胞内运输。因此，这些粒子可以用来向细胞表面和内部传递信息。为什么是化学和基因信号？使用干细胞再生组织的关键是能够说服它们形成所需的类型。有两种方法可以操纵这些细胞形成骨/直接对内部细胞核进行遗传修饰，或使用间接的外部刺激，如其他细胞分泌的化学物质或存在于局部细胞环境中，以及与其他细胞的物理接触。为什么是3D涂层？骨样细胞行为是通过向培养基中添加昂贵的化学物质或将其吸附到基质上间接诱导的。直接的基因改造需要将DNA传递到细胞核，通常是通过附着在小的载体颗粒上。例如，使用HAP传递基因的标准方法包括将前体化学物质混合在一起，然后让形成的晶体像暴风雪一样随机落在细胞上。造成目前这两种方法效率低下的主要因素有两个；(a)细胞粘附在底物上，所以不是所有的细胞表面都可用，(b)细胞本身不是靶向的，所以添加剂的浓度比必要的要高。这两个问题都可以通过我们提出的方法得到缓解。

Our aims are to develop new, improved materials and methods which will allow stem cells (mesenchymal; MSCs) to be manipulated to form bone tissue. We will do this using tiny (<100 nm; 1/1000 diameter human hair) calcium phosphate (hydroxyapatite; HAP) particles as vectors to carry specific biological molecules to the cells. To maximize the delivery of these chemical and genetic signals, the whole cell surface of each individual cell will be covered with the vectors (3D coating). Specifically, this will allow us to produce and grow self supporting, living bone tissue, either inside the body at the site of damage, or outside in culture dishes ready for implant. More generally, the improved efficiency and cost effectiveness of this approach will also enhance studies in the generation of other tissue types from MSCs (e.g. nerve and muscle) and in modifying other types of stem cell.Why mesenchymal stem cells?Stem cells have huge potential as therapeutic agents. Embryonic stem cells (ESC) have the potential to form all the major types of cell in the body, and are relatively easy to grow in culture. However, there are ethical and compatibility concerns with there use. Adult stem cells (ASC) can be harvested from specific tissue types (blood, nerves, skin), but the populations need to be expanded to get sufficient material for therapeutic use. In this regard, bone marrow mesenchymal stem cells (MSC) offer great hope for tissue engineering as methods for isolation and rapid cultivation are well established. They are natural precursors to bone, cartilage, fat and fibrous connective tissue formation. Thus they are already intensively studied as components of systems for replacing damaged bone tissues (e.g. restorative surgery). In addition the same person can be donor and recipient, thus alleviating the problems associated with ESCs.Why small hydroxyapatite particles?Hydroxyapatite is the chemical form of calcium phosphate found in bone, so it is compatible with the cells. The crystals of HAP in bone are also of a similar size (<100 nm). In addition, because the crystals are so small as well as coating the cell, some will be transported inside the cell. Thus the particles can be used to deliver information to the cell surface and interior.Why chemical and genetic signals?The key to using stem cells to regenerate tissue is the ability to persuade them to form the required type. There are two ways to manipulate these cells towards bone formation / direct genetic modification of the internal cell nucleus, or the use of indirect external stimuli such as chemicals secreted by other cells or present in the local cell environment, and physical contact with other cells.Why 3D coating?Bone-like cell behaviour is induced indirectly by adding expensive chemicals to the culture medium or adsorbing them onto the substrates. Direct genetic modification requires DNA to be delivered to the cell nucleus, typically by attachment to small carrier particles. For example, standard methods for gene delivery using HAP involve mixing the precursor chemicals together, then allowing the crystals that form to randomly settle on the cells like a snowstorm. There are two main factors which contribute to the inefficiency of both these current approaches; (a) the cells are adhered to a substrate, so not all the cell surface is available, and (b) the cells themselves are not targeted, so the additives are used at higher concentration than necessary. Both these problems should be alleviated by our proposed method.