Institut für chirurgische Forschung und Spitalmanagemement

Mrs. Simone Kramer
PhD Commission
Faculty of Medicine, University Basel
Klingelbergstr. 61
CH - 4056 Basel

Basel, 26thFebruary 2013

Progress Report of Dr. Celeste Scotti, PhD Candidate at the Institute of Surgical Research and Hospital Management (ICFS), Tissue Engineering Laboratory, University Hospital Basel.

Evaluation of the 2nd year (PhD committee meeting: February 12, 2013)

Dear members of the PhD-commission,

I started my PhD studies at the Institute of Surgical Research and Hospital Managment (ICFS) of the University Hospital of Basel on January 2, 2011 after finishing my Residency in Orthopaedic Surgery by the University of Milan, Italy in November 2010. This progress report takes into account the first two years of research performed to date.

My PhD Project is focused on the development of a clinically-relevant bone substitute with the features of a “bone organ” through endochondral ossification. These two years were dedicated to the development of an upscaled model of endochondral ossification with bone marrow-derived human adult mesenchymal stromal cells, to the further improvement of this model and to the study of possible hematologic applications of this model. The latter lead to the publication of a paper on PNAS in January 2013. This work is also in continuity with the work I performed in the same laboratory, always under the guidance of Prof. Martin and PD Dr. Barbero, during a 15-month visiting fellowship between June 2008 and August 2009.

My career goal is to become a physician scientist involved in Orthopaedic Surgery and Regenerative Medicine. For this reason, in parallel with my research work in these two years, I attended the Trauma Clinic (Prof. M. Jakob) of BaselUniversityHospital and I was the recipient of a Travelling Fellowship by the European Society of Knee Surgery Sport Traumatology Artroscopy (ESSKA). During this fellowship I attended 8 scientific seminars, gave 8 talks (3 different topics) and attended an international meeting (German-Speaking Association of Arthroscopy –AGA- Regensburg, 2011). The work performed to prepare these talks and to attend the seminars and the meeting correspondsto 2 ECTS (3 hours per seminar, 10 hours to prepare each of the 3 talks). I would kindly ask that these efforts are converted in ECTS.

The 3rd year of PhD studies will be conducted abroad, at the Galeazzi Orthopaedic Institute (Milan, Italy) in the framework of a European collaborative project (ENDOMATRIX - Eurostars Programme) that has been granted also thanks to the results of this PhD project. During this year, I won’t be able to attend ECTS course in Basel. In order to compensate, to properly continue my training and to reach the total of 12 ECTS by the end of my third year of studies, I will attend ECTS courses for PhD students by the University of Milan.

In the following, I will summarize the aims and main achievements of these first two years.

Original Research Plan Summary

  • Aim 1: Identifiy conditions for reproducible hypertrophic differentiation by Mesenchymal Stromal Cells(Aim 1a) and test osteoinductivity of devitalized grafts (Aim 1b).

Hypothesis:

Aim 1a. Culture conditions for generating hypertrophic cartilage can modulate in vitro tissue formation.

Aim 1b. Devitalized hypertrophic cartilage templates can be generated with Mesenchymal Stromal Cells (MSC) and are osteoinductive. In particular, they have the potential to regenerate bone through endochondral ossification by recruiting osteoprogenitors, vasculature and osteoclasts and being thus remodeled into bone.

Experimental Approach:

Aim 1a. To address this question, we explored both a “Developmental Engineering” approach, by stimulating the Indian Hedgehog (IHH) pathway, and a “Biomimetic” approach, by adding Interleukin-1β (IL-1β) in order to simulate the environment of fracture repair. IHH is required for the endochondral process of hMSC (Scotti C et al. PNAS, 2010). We, therefore, evaluated the effect of Purmorphamine, a small molecule activating the IHH pathway on the in vitro development of the hypertrophic cartilaginous engineered tissues. Purmorphamine administration for up to the first 7 days of culture resulted in cell condensation and upregulation of BMPs and IHH genes. However, a short exposure to Purmorphamine resulted in a higher BMPs expression and better tissue formation. IL-1β, together with TNF-α, is the main inflammatory chemokine involved in fracture repair. We found that in our model, IL-1β increases the total calcium content of our engineered tissues and overall speeds-up the remodeling process of the cartilage template into bone, thus leading to a more efficient endochondral bone formation.

Aim 1b. To address the second part of this Aim, we devitalized hyperthrophic cartilage template and assessed their endochondral bone formation potential. Overall, the freeze/thaw procedure led to a 60% loss of ECM components such as GAG or calcium and of key factors for the endochondral route such as MMP-13, VEGF and BMP-2. This loss in bioactive molecules resulted in a much less efficient bone formation compared to vital controls. This finding highlighted the importance of preserving the ECM of the tissue in our model. We, therefore, abandoned this simplistic approach and we are now evaluating alternative strategies to a more efficient devitalization method.

Outcome:

This part of the project is concluded. These experimentslead to an optimization of the in vitro parameter for engineering endochondral constructs based on the assumption that our model resembles more bone healing rather bone formation and therefore benefits from IL-1β (Mumme, Scotti et al, eCM, 2012). The part on devitalization will now be used as starting point for further studies, outside the frame of this PhD project), focused on developing alternative and more efficient methods for tissue devitalziation.

  • Aim 2: Scale-up tissues to a clinically relevant size using 3D porous scaffolds and a perfusion-based bioreactor system and assess bone forming potential of upscaled tissue.

Hypothesis:

Hypertrophic cartilage templates can be upscaled to clinically-relevant size with use of 3D porous scaffold and perfusion bioreactors and maintain the bone generation potential of the previously developed small-scale model.

Experimental Approach:

The first strategy we used to scale up the engineered endochondral tissues consisted in culturing hMSC statically on 3D collagen scaffold using the same culture condition previously identified (Scotti et al. PNAS, 2010) and a protocol for upscaled engineered cartilage based on a 8mm diameter collagen scaffold (UltrafoamTM, Davol, USA) and high-cell density (Scotti C, TE-A, 2012). The resulting upscaled engineered endochondral samples displayed the same in vitro development of the small-scale with formation of a peripheral “bone collar” rich in collagen type I and Bone Sialoprotein.We also assessed their endochondral bone forming potential by implanting them ectopically in nude mice. The grafted up-scaled tissues after 12 weeks in culture in such in vivo environment generated bone tissue with the features of a “bone organ” rich in bone marrow, vessels, osteogenic cells, bone extracellular matrix (ECM) and osteoclasts. Bone marrow has been extracted and characterized for phenotype with flow cytometry showing presence of LSK cells (suggesting presence of haematopoietic stem cells).

In particular, we demonstrated that the marrow component of the engineered bone organ, occupying the majority of its volume, includes phenotypically and functionally defined HSC at a comparable frequency than normal bones of the same mice.These unprecedented findings validate the physiological nature of the in vivo established ossicle, and reinforce the evidence of self-organization ability of hMSC-based hypertrophic cartilage templates into functional hematopoietic niches (Scotti, PNAS 2013).

The aforementioned static model serves as a basis for the next step of the project in which we will develop a perfusion-based bioreactor culture protocol using Ultrafoam collagen scaffolds of the same size as in static culture. Perfusion has been performed through the use of a U-tube culture system with 2.7 ml/min flow during seeding and 0.27 ml/min flow during the rest of the culture. Under these conditions homogenous tissue with a good glycosaminoglycan content has formed after 3 weeks of chondrogenic culture. In vivo implantation in nude mice demonstrated a comparable bone forming efficiency to that of samples engineered in static conditions, paving the way to the application of automated culture systems for manufacturing upscaled endochondral matrices (manuscript in preparation).

Expected Outcome:

This upscaled endochondral bone model presents the features of an ectopic “bone organ” rather just “bone tissue”. For this reason it will be exploited in several studies ranging from regenerative medicine to hematology and oncology. Consistently with respect to the original plan, in the next year we will focus on the use of endochondral matrices in orthotopic bone-defects models in rats and rabbits to evaluate the clinical relevance of this model.

Progress according to time-table

Aim 1

The time schedule was respected as initially planned:

  • Culture conditions which can improve the reproducibility of endochondral bone formation have been identified.
  • Preliminary assessment of devitalized construct has been performed and alternative techniques will be developed according to the results obtained to date.
  • This aim can be considered achieved and the work led to a published manuscript.

Aim 2

The time schedule was respected as initially planned:

  • The upscaled model of engineered cartilage led to a published paper (Scotti C, et al. TE-A, 2012)
  • The model developed for this Aim brought to unexpected results in terms of biologic relevance and possible exploitations. Reproducing an ectopic “bone organ” will allow us to explore the interplay between the graft and the host in term of bone formation, bone marrow engraftment and, possibly, mechanisms of cancer metastatization. Importantly, this will result in a broadening of the perspectives of this project in the next years.
  • A landmark paper demonstrating the development of a “bone organ” model, according to a developmental engineering paradigm has been published (Scotti, PNAS, 2013).
  • Bioreactor-based culture and alternative devitalization technique will be further developed during the next year of studies.

Aim 3

  • This part of the project will be developed during the 3rd year of project and preliminary studies towards this goal started already.

Training and awards of the first two years

1)Acquiring cell cultures techniques for human nasal/articular chndrocytes and bone marrow-derived mesenchymal stromal cells.

2)Acquiring tissue engineering techniques for construct production.

3)Acquiring basic techniques for 3D perfusion culturing, differentiation and proliferation techniques in a bioreactor system.

4)Acquiring the techniques for quantitative and qualitative tissue analyses with optic, fluorescence and confocal microscopy (immunohistochemistry, histology, immunofluorescence).

5)Acquiring the techniques for biochemical analysis of tissue (DNA and GAG measurement, ELISA).

6)Acquiring the techniques for qPCR.

7)Recipient of the European Arthroscopy Travelling Fellowship of the European Society of Knee Surgery Sport Traumatology Artroscopy (ESSKA).

8)Robert Mathys Student Award for the best oral presentation at the „XIII eCM Conference: Bone Fixation, Repair & Regeneration“ (2012)

9)Travel Grant Award for the 2012 10th annual ISSCR Meeting

10)Travel Grant Award al TERMIS World Congress, Vienna, 5-8 settembre 2012

11)Basic science award at the 39th Annual Meeting of the European Group forBlood and Marrow Transplantation (2013)

12)Best Poster at the Annual meeting of the Swiss Stem Cell Network (2013)

Posters and Presentations on PhD topic

1)Developmental Engineering of a Bone Organ”; TERMIS World Congress, Vienna, 5-8 september 2012- PRESENTATION

2)“Engineering a bone organ’’; XIII eCM Conference: Bone Fixation, Repair & Regeneration –Davos, June 2012 - PRESENTATION

3)’’Developmental engineering of a functional HSC niche through endochondral ossification” Annual Meeting of the International Stem Cell Research Society (ISSCR) – June 2012 – POSTER

4)“Advances in Bone Regeneration”. Orthopaedic Residents Training Program, Galeazzi Orthopaedic Institute, Milan. INVITED LECTURE.

Publications related to the PhD Project

1)Scotti C, Leumann A, Candrian C, Barbero A, Croci D, Schaefer DJ, Jakob M, Valderrabano V, Martin I. Autologous tissue-engineered osteochondral graft for talus osteochondral lesions: state of the art and future perspectives. Techniques in Foot and Ankle Surgery – 10(4):163-168.

2)Scotti C, Osmokrovic A, Wolf F, Miot S, Peretti GM, Barbero A, Martin I. Response of human engineered cartilage based on articular or nasal chondrocytes to il-1β and low oxygen. Tissue Engineering A 2012 Feb;18(3-4):362-72

3)Jakob M, Saxer F, Scotti C, Schreiner S, Studer P, Scherberich A, Heberer M,Martin I. Perspective on the evolution of cell-based bone tissue engineeringstrategies. Eur Surg Res. 2012;49(1):1-7.

4)Mumme M*, Scotti C*, Papadimitropoulos A, Todorov A, Hoffmann W, Bocelli-Tyndall C, Jakob M, Wendt D, Martin I, Barbero A. Interleukin-1β modulates endochondral ossification by human adult bone marrow stromal cells. Eur Cell Mater. 2012 Sep 24;24:224-36. *co-first author

5)Scotti C, Piccinini E, Takizawa H, Todorov A, Bourgine P, Papadimitropoulos A, Barbero A, Manz MG, Martin I. Engineering of a functional bone organ through endochondral ossification. Proc Natl Acad Sci U S A. 2013 Feb 11.

6)Status: Manuscript in preparation Tentative title: Regenerating bone through devitalized hypertrophic cartilage templates.

Submission: End of 2013

7)Status: Manuscript in preparation Tentative title: Engineering upscaled endochondral tissues with perfusion bioreactor system.

Submission: End of 2013

ECTS Lectures to date

University of Basel

1)Tissue engineering symposium: 2011 – present (3 credit points)

2)Regenerative Medicine Journal Club: 2011 – present (3 credit points)

3)Biomechanik, Bewegungsanalyse, Biokalorimetrie und klinische Forschung: 2012 (1 credit point)

International Meetings attended during 2012

TERMIS World Congress - Vienna, Austria

International Congress on Stem Cell Research (ISSCR) – Yokohama, Japan

European Cells and Materials – Davos, Switzerland

Feedback from the doctoral committee

Prof. Dr. Ivan Martin:

In the last year, Dr Celeste Scotti demonstrated a high motivation and committment to pursue research, being also capable to independently set and address relevant scientific targets. His training as certified Orthopaedic Surgeon and the previous 15-month research experience in my lab gave him a unique background for Regenerative Medicine studies and the potential to work as a bridge between Clinic and Research. His work on bone regeneration gave a unique contribution tot he field and resulted in high-level scientific publications on top Journals (PNAS). I’m confident Dr Celeste Scotti will succeed in finalizing his work and generatefurther publications as planned.

PD. Dr. Andrea Barbero:

Dr Celeste Scotti previously worked with me during a visiting fellowship in our lab from june 2008 to august 2009. In the last year, he acquired scientific maturity, capability of independently plan and organize scientific experiments, very good skills in scientific writing, knowledge and technical skills in MSC biology and Tissue Engineering.He was able to reach the planned objectives and to acquire good knowledge and skills in such competitive fields. I am confident that Dr Celeste Scotti will be able to successfully continue his work and publish in high impact factor journals.

Personal data and project data

PhD student: Scotti, Celeste (Student Number10-070-092)

PhD Subject: Developmental Engineering of a bone organ through endochondral ossification (working title)

Thesis committee (Dissertationskomitee):

  • Prof. Dr. Ivan Martin (Fakultätsverantwortlicher)
  • PD. Dr. Andrea Barbero (Dissertationsleitung)

Yours sincerely,

Dr. med. Celeste Scotti