Regeneration of tissue interfaces: Biomaterial controlled morphogen gradients for tissue patterning in regeneration

骨软骨缺损的愈合对骨科医生来说仍然是一个挑战，目前没有一种技术能够实现骨和软骨阶段的完全重建和功能。临床治疗成功有限的一个原因是骨软骨连接处的骨和软骨组织紧密相互作用，这是一种典型的独特组织模式。在这个项目中，我们希望以骨软骨缺损为例，验证再生医学中不同组织模式的新概念。我们的概念基于发育生物学的基本原理，其中包括局部激活剂和抑制剂紧密结合在一起形成不同的组织模式。为此，我们合作开发了一种多层结构（基于微球的PLG支架），可以控制刺激剂及其拮抗剂从不同层的释放。我们计划利用我们的生物力学控制良好的羊骨软骨缺损模型在体内验证这一概念，并与内源性因子释放进行比较。具体来说，层状支架由含有tgf β和抗BMP4的软骨区、含有抗bmp和抗tgf β抗体的中间缓冲带和由BMP4和抗tgf β组成的成骨诱导层组成。我们的假设是，PLG支架在空间上控制刺激因子和对立因子的释放，可以在羊骨软骨缺损模型中引导组织模式（软骨样组织-时间标记-软骨下骨）。我们分析的基础是通过早期血肿形成来评估骨软骨缺损的内源性反应。为了实现这一目标，我们将研究在植入不含生长因子或仅含TGFbeta3或BMP4的支架后的第一个小时内启动的炎症反应级联。我们将主要研究细胞因子模式和炎症细胞对新骨软骨缺损血肿的反应。下一步，我们将通过植入加载TGFbeta或BMP-4的支架来分析TGFbeta和BMP-4对这一阶段的影响。总之，所提出的分析将允许判断基于生物材料的策略的可行性，以控制不同组织连接处的局部组织形成，如骨软骨修复。此外，这将有助于了解这种方法是否有助于克服目前生长因子tgf - β和BMP4在骨软骨修复中的临床缺陷。一旦证实，这种不同组织形成的概念可以应用于多个其他甚至更复杂的器官结构。

The healing of ostechondral defects in the joint still remains a challenge for orthopaedic surgeons and none of the current techniques achieve full reconstruction and functionality of the bone and cartilage phases. A reason for the limited clinical success of treatments is the tight interplay of bone and cartilage tissues at the osteochondral junction, a classical distinct tissue pattern. With the proposed project we want to validate a new concept for distinct tissue patterning in regenerative medicine using osteochondral defects as an example system. We base our concept on a fundamental principle of developmental biology, which includes locally activators and inhibitors acting tightly together to form distinct tissue patterns. For that purpose a multi-layered construct (microsphere-based PLG scaffold) that allows for the controlled release of both stimulatory agents and their antagonists from distinct layers ws developed in cooperation. We plan to employ our biomechanically well controlled osteochondral defect model in sheep to validate the concept in vivo and compare to endogenous factor release. Specifically, the layered scaffold is composed of a cartilage zone containing TGFbeta and anti-BMP4, a middle buffer zone containing anti-BMP and anti-TGFbeta antibodies and an osteogenic inductive layer composed of BMP4 and anti-TGFbeta It is our hypothesis that spatially controlled release of both stimulatory and opposing factors from the PLG scaffold can steer tissue patterning (cartilage like tissue-tidemark-subchondral bone) in an osteochondral defect model in sheep. Basis to our analyses will be the evaluation of the endogenous response to an osteochondral defect by means of the early hematoma formation. Towards that aim, we will investigate the inflammatory response cascade initiated within the first hours after implantation of a scaffold containing either no growth factors or TGFbeta3 or BMP4 alone. We will primarily investigate the cytokine pattern and inflammatory cell response in the hematoma that develops in the freshly created osteochondral defect. In the next step, the suggestibility of this phase by TGFbeta and BMP-4 will be analyzed by implanting the scaffold loaded either with TGFbeta or BMP-4. In summary, the proposed analyses will allow judging on the feasibility of a biomaterial-based strategy to control locally tissue formation at distinct tissue junctions such as in osteochondral repair. Further, it will allow gaining understanding if such approach is helpful to overcome the current clinical shortcomings in osteochondral repair by the growth factors TGFbeta and BMP4. Once validated, this concept of distinct tissue formation may be applied to multiple other even more complex organ structures.