Regenerative Performance and Immunogenicity of Engineered Extracellular Matrices

What have your Ph.D. studies focused on?

“Bone is a highly dynamic organ central to movement, mineral homeostasis, and structural integrity, and one of the few tissues capable of scar-free repair; however, intrinsic healing is often insufficient to restore critical-sized defects that arise after trauma, tumor resection, or congenital malformations. Despite advances, existing treatments remain limited in terms of reliability and scalability, underscoring the need for safer, more effective, and customizable off-the-shelf strategies. 

Developmental engineering provides such a strategy: instead of forcing repair with mature cell and tissue components, it designs grafts that recapitulate embryonic morphogenetic programs to guide regeneration. In the skeleton, the relevant process is endochondral ossification, which forms most of our bones and is also a natural repair mechanism. The physiological sequence in which a cartilage template forms, undergoes controlled hypertrophy, recruits vasculature and osteoprogenitors, and is remodelled into bone. Harnessing this conserved pathway offers a principled route to robust, vascularised bone formation in vivo.

In the laboratory of Cell, Tissue and Organ engineering , our focus is on uncovering the mechanism of bone formation to design effective therapies. Within this context, my PhD research specifically focuses on extracellular matrix (ECM) based scaffolds as developmental templates for bone regeneration. The ECM is a complex, noncellular network of proteins and other molecules, in the body it provides support and structure to cells in tissues and organs.

Building on our previous work demonstrating that custom human mesenchymal stromal cell (hMSC) lines can be used to manufacture engineered ECMs (eECMs), the first part of my thesis evaluated the integration, regenerative potential, and immune responses of devitalized, cartilage-derived eECM grafts across multiple in vivo models. The grafts consistently demonstrated robust osteoinduction, new bone formation, supporting their potential as off-the-shelf scaffolds. A significant component of this work profiled macrophage recruitment and polarization. Macrophages are a group of immune cells that destroy germs and damaged cells and promote tissue repair.  Here, we identified correlations between early M2 macrophage infiltration and successful regeneration, emphasizing the immune system’s role as a determinant of graft performance and suggesting that engineered ECMs can be designed to engage immune responses constructively. 

Guided by these findings, the second part of my thesis investigated whether ECM composition could be intentionally tailored using genetic approaches, primarily CRISPR/Cas9. To our knowledge at the time, this approach had not been applied to the compositional modification of eECMs. We modified hMSC lines to knock out two genes: VEGF, coding for proteins promoting the formation of new blood vessels, and RUNX2, a master regulator of bone development . We then assessed their ability to generate cartilage matrices with distinct osteoinductive capacity.

Our experiments successfully knocked out VEGF and RUNX2 in engineered cartilage tissues using CRISPR/Cas9, revealing nuanced roles in cartilage formation and bone regeneration. Despite VEGF knockout, the engineered cartilage retained its capacity to undergo endochondral ossification, indicating VEGF might not be essential for initiating this process. In contrast, RUNX2 knockout affected the graft composition, exhibiting reduced collagen X content (a hallmark of hypertrophy). This led to a delayed remodeling into bone, thus better preserving the original cartilage template. Here, the RUNX2 strategy bears relevance for cartilage repair, as demonstrated in an osteochondral defect models in rats.

In summary, we demonstrated the performance of ECMs in instructing tissue repair using standardized cell lines, of which genetic customization results in tailored graft properties. We assessed the immunogenicity of ECMs and identified early immune response patterns in engineered ECMs linked to successful tissue regeneration. I firmly believe this work addresses a key obstacle in regenerative medicine and provides a clear pathway toward clinically translatable, personalized implants with potential applications beyond skeletal repair.”

Can you tell us more about the cover of your thesis?

“My aim was to design a cover that depicts the heart of my thesis, as my title suggests: the regenerative performance and immunogenicity of engineered extracellular matrices. Since my artistic range is, let's say, aspirational, I enlisted my potential future replacement: AI (ChatGPT, to be specific). I created a prompt to visualize osteogenesis as a kind of sci-fi assembly line, with immune cells, growth factors, and vessels flowing together to form new bone. I requested it in a Van Gogh style, partly because I admire his work, but mainly because those swirling strokes and bold contrasts mirror the energy of cellular crosstalk and vascular ingrowth. So, beyond being visually striking, I also want this cover to reflect my doctoral work, produced not alone but through the contributions of many.”

How did you end up doing a Ph.D. at Lund University and the Lund Stem Cell Center?

“I am from India. During my bachelor's studies, I chose Biotechnology because I thought it could serve as a bridge between biology and engineering. Little did I know back then that I would end up doing my masters in Germany in Biochemical engineering. 

During my master’s thesis, I studied the role of non-coding RNA in dendritic cell differentiation. It was the first time I truly understood how stem cells are programmed and specialized and how many factors, from genetic to post-translational, can influence that process. It also revealed the complexity and layering of the immune system, as well as the extent to which our immune memory can become. I was fortunate to work with the CRISPR/Cas9 technology, one of the most revolutionary technologies in life sciences.

I wanted to continue my research, so when I found an opportunity to work with immune cells and bones (their origin) using CRISPR/Cas9, I applied immediately. The chance to start my doctoral work in a new lab was exciting. I enjoyed the challenge of setting up almost everything from scratch. Additionally, pursuing a PhD in Sweden, with its stunning natural landscapes, international atmosphere, and top-tier facilities, felt like a real bonus.”

What have you found the most enjoyable during your Ph.D. studies?

“There are several aspects I enjoyed at different points during my thesis. I truly appreciated the international and multidisciplinary environment at Lund University. Working with scientists from diverse backgrounds gave me entirely different perspectives, both scientific and personal. 

I also enjoyed the entire research process, from formulating a hypothesis, reading the literature, planning experiments, learning new techniques, obtaining results, and interpreting the data. I believe my thesis work can make a direct impact because it addresses current treatment methods while developing a new approach to overcome their limitations. I also valued the flexibility to work longer when an experiment needed more time and to take a lighter day afterward. 

Besides my main projects, the research school and MentLife provided valuable training in interdisciplinary work, PI perspectives, industry, and grant and article writing. Overall, these experiences prepared me not only for the PhD but also for my broader scientific career.”

What has been the most challenging aspect?

“One of the toughest challenges I've spent the most time on was decision-making. It involved choosing which experiment or project to prioritize, managing my time effectively, knowing when to push hard, and deciding when to move on if experiments didn’t go as planned. I can’t say I’ve completely solved it, but I firmly believe I’ve become much better at it.”

What are your plans following your Ph.D. defense?

“I am in a unique situation. My PhD contract ended nearly two years ago, and I immediately started working at a different lab as a research engineer. Currently, I work in Sandra Lindstedt's Lab, which is also part of the Lund Stem Cell Center, mainly as a molecular biologist focusing on lung gene therapy. I plan to stay on as a postdoctoral researcher in the same lab after my defense.”

Any tips or advice for future Ph.D. students?

“I believe everyone has their own way of doing things. I learned a few lessons the hard way, especially during the later stages of my PhD. I lacked confidence when expressing my ideas or opinions, so I recommend being more confident and not hesitating to ask for help. From what I have seen, almost everyone is willing to help if asked. I struggled to keep proper records of my work and was a bit disorganized, so I suggest keeping organized notes, from protocols to to-do lists. And I always try to remember that, better late than never, we can always start over.”