Einleitung: Degenerativ-entzündliche Gelenkerkrankungen und insbesondere die Osteoarthritis (OA) gehören zu den häufigsten orthopädischen Erkrankungen beim Pferd und führen in vielen Fällen zu langfristigen und teilweise dauerhaften Leistungseinschränkungen. Ätiologisch ist die OA bei jungen Pferden hauptsächlich auf ein akutes oder repetitives Trauma zurückzuführen, während bei älteren Pferden von einem chronisch-degenerativen Krankheitsprozess ausgegangen werden kann. Zahlreiche klinische Studien konnten vielversprechende Therapieerfolge mit der intraartikulären Anwendung von potenziell regenerativ wirkenden Therapeutika aus autologen und allogenen Blutprodukten oder mesenchymalen Stromazellen (MSC) in unterschiedlichen OA-Modellen darlegen. Allerdings besteht aufgrund der heterogenen Studienlage bisher weitgehend Unklarheit über den Langzeiterfolg dieser sogenannten Orthobiologika bei Pferden mit natürlich auftretender OA. Zielsetzung: Die Zielsetzung dieser Arbeit war es, über eine statistische Aufarbeitung der Studienlage aus den Jahren 2007-2020 eine fundierte Aussage zu Langzeiterfolgen und zur Anwendungssicherheit von intraartikulär applizierten Orthobiologika bei natürlich auftretender OA beim Pferd zu treffen. Es wurde die Hypothese aufgestellt, dass die Anwendung von Orthobiologika eine sichere und effektive Therapieoption bei Pferden mit natürlich auftretender OA ist. Methoden: Für die Beurteilung wurde ein systematisches Review der aktuell publizierten Literatur in englischer und deutscher Sprache der Jahre 2007 bis 2020 mit anschließender Meta-Analyse angefertigt und ausgewertet. Die Literaturrecherche erfolgte über öffentlich zugängliche, anerkannte Wissenschaftsplattformen unter Nutzung definierter Schlüsselwörter. Es wurden zunächst alle klinischen Studien, die sich mit der Anwendung von Orthobiologika bei natürlich entstandener OA beim Pferd auseinandersetzen gesammelt und entsprechend festgelegter Ein- und Ausschlusskriterien selektiert. Für das systematische Review wurden klinische Studien, die eine intraartikuläre Behandlung mit MSC und/oder autologen Blutprodukten mit einer sechsmonatigen oder längeren Nachuntersuchungszeit der Pferde erfasst. Verglichen wurde der Lahmheitsgrad, gemessen an den Erfolgen in Wettkämpfen, Rückkehr in den Turniersport oder der Nutzung auf dem ursprünglichen Leistungsniveau, vor und nach der intraartikulären Behandlung. Zudem wurden auftretende Nebenwirkungen nach der Behandlung dokumentiert. Für die Meta-Analyse wurden ausschließlich randomisierte und kontrollierte Studien (RCTs) genutzt. Exkludiert wurden in-vivo-Studien mit induzierter OA, invitro- Studien oder Studien mit konventionellen Behandlungsmethoden. Jede Studie des systematischen Reviews wurde auf systematische Fehler untersucht und auf das Biasrisiko gemäß den „Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)“-Leitlinien getestet. Um die Ergebnisse mittels der Methodik der Meta-Analyse zu vergleichen, wurde das Quotenverhältnis (Odds Ratio) mit einem 95 % Konfidenzintervall berechnet. In einem random-effects Modell wurde jede Studie mit ihrer geschätzten Effektgröße und dem entsprechenden Konfidenzintervall im Forest Plot grafisch dargestellt. Die Heterogenität wurde mit I² für die Studien berechnet und mit einem Ergebnis von I² > 50 % als signifikant eingestuft. Ein Ergebnis wurde als signifikant angesehen, wenn sich p < 0,05 darstellte. Ergebnisse: Die elektronische und manuelle Recherche ergab insgesamt 271 Studien. Davon erfüllten 13 Studien die Einschlusskriterien für das systematische Review, wobei in fünf Studien die OA mit MSC behandeltet wurde. In acht der 13 Studien wurden autologe Blutprodukte verabreicht, davon wurde in drei Studien autologes konditioniertes Serum (ACS) angewandt. In den verbleibenden fünf Studien wurden zelluläre Eigenblutprodukte mit einer erhöhten Thrombozytenkonzentration verwendet. Insgesamt wurden bei zwei der 13 Studien ein geringes Risiko für eine statistische Verzerrung festgestellt. Nach der intraartikulären Behandlung mit orthobiologischen Therapeutika wurde eine durchschnittliche Verbesserung des Lahmheitsgrades um 65 % erreicht, unabhängig von der Art des verwendeten Orthobiologikums. Elf der 13 Studien zeigten einen generellen positiven Effekt nach der Behandlung, mit Pferden die auf ihr ursprüngliches Leistungsniveau zurückkehrten. Vier der 13 Studien erwiesen sich geeignet für die Meta-Analyse und wurden mittels Forest Plot exprimiert. Hier wurde die Reduktion des Lahmheitsgrades der Behandlungs- und der Kontrollgruppe verglichen. Die Heterogenität der Langzeitstudien mit einem Beobachtungszeitraum von mindestens sechs Monaten war moderat mit einem Heterogenitätsindex von I² = 55 %. Alle Studien mit einem OR-Wert > 1 begünstigen die experimentelle Gruppe, was hier zutreffend war (OR 17,02; 95 % CI: 8,5474 bis 33,8849 p < 0,0001). In keiner Studie wurde die Grenze zur Unwirksamkeit überschritten, was darauf hindeutet, dass der Behandlungseffekt in allen Studien als ähnlich eingeschätzt wurde. In der Langzeitbeobachtung führte die Anwendung von intraartikulär verabreichten orthobiologischen Therapeutika zu einer Verringerung der Lahmheit bei 73 %, während die Lahmheit in der Kontrollgruppe bei 17 % reduziert wurde. Transiente Nebenwirkungen im Sinne eines sogenannten „joint flare“, einer aseptischen, entzündlichen Gelenkreaktion, waren in allen dokumentierten Fällen (n = 15, 3 %) der insgesamt 518 ausgewerteten Pferde überwiegend selbstlimitierend und führten in nur einem Fall aufgrund fehlender Aufrechterhaltung der Verblindung zu einem Studienausschluss. Schlussfolgerung: Den eingeschlossenen Studien zufolge zeigten Pferde mit natürlich auftretender OA nach einer intraartikulären Behandlung mit orthobiologischen Therapeutika im Vergleich zur Kontrollgruppe im Langzeit-Follow-up einen signifikant geringeren Grad an Lahmheit bei hoher Anwendungssicherheit. Keines der Pferde zeigte nach Behandlung signifikante Nebenwirkungen.:Inhaltsverzeichnis Abbildungsverzeichnis Abkürzungsverzeichnis Tabellenverzeichnis 1. Einleitung 2. Literaturübersicht 2.1 Die Osteoarthritis beim Pferd 2.1.1 Ätiologie beim Pferd 2.1.2 Pathomechanismus der Osteoarthritis 2.1.3 Osteoarthritis auf zellulärer Ebene 2.1.4 Histologische Studien zur Osteoarthritis beim Pferd 2.2 Regenerative/-Orthobiologische Therapien bei Osteoarthritis 2.2.1 Das therapeutische Konzept orthobiologischer Therapieansätze 2.2.2 Behandlungsoptionen der Osteoarthritis beim Pferd 2.2.3 Osteoarthritis: Das Pferd als Modelltier 2.3 Evidenzbasierte Medizin 2.3.1 Definition der evidenzbasierten Medizin 2.3.2 Das Systematische Review 2.3.3 Risk of bias 2.3.4 Die Meta-Analyse 3. Publikation 4. Diskussion 4.1 Methodische Diskussion der Studienauswertung 4.2 Kritische Begutachtung verschiedener OA-Modelle 4.3 Heterogenität der Studien 4.3.1 Diversität der Behandlungsmethoden 4.3.2 Uneinheitliche Definition des Erfolgsmerkmals und der Untersuchung 4.4 Kritik an randomisierten, kontrollierten Studien 4.5 Kritik an der Meta-Analyse 4.6 Schlussfolgerung 5. Zusammenfassung 6. Summary Literaturverzeichnis Danksagung / Introduction: Degenerative-inflammatory joint diseases, specifically osteoarthritis (OA), are among the most common orthopedic diseases in horses and usually lead to long-term and even permanent performance limitations. Etiologically, OA in young horses is mainly caused by an acute or repetitive joint trauma, whereas chronic degenerative process is stated in older horses. Numerous clinical studies have been demonstrated promising therapeutic success by intra-articular application of potentially regenerative therapeutics derived from autologous and allogeneic blood products or mesenchymal stromal cells (MSCs) in different OA models. However, the long-term success of these so named orthobiologics in horses with naturally occurring OA is controversially discussed due to the heterogeneity of studies. Objectives: The objective of this study was a statistical analysis of published literature between the years 2007-2020 regarding long-term success and safety of intraarticular orthobiologics in naturally occurring OA in horses. It was hypothesized that the use of orthobiologics is a safe and effective therapeutic option in horses with naturally occurring OA. Material and Methods: For assessment, a systematic review of the currently published literature in English and German from 2007-2020 with subsequent meta-analysis was performed and evaluated. The literature search was conducted via publicly available, recognized scientific platforms using defined keywords. First, all clinical studies dealing with the use of orthobiologics in naturally occurring OA in horses were reviewed and selected according to defined inclusion and exclusion criteria. All clinical trials that included intraarticular treatment with MSCs and/or autologous blood products with an at least six-month follow-up were included in the systematic review. The degree of lameness prior and after intraarticular treatment was evaluated (comparison of the success rate, horses working on competition, horses working at trainings level, lame free horses). In addition, any adverse effects that occurred after treatment were documented. For meta-analysis only randomized controlled trials (RCTs) were included. In vivo studies with chemically or experimentally induced OA, in vitro studies or studies used conventional treatments were excluded. Each study included in the systematic review was examined to the risk of bias according to the „preferred reporting items for systematic reviews and meta-analyses (PRISMA)” guidelines. To compare dichotomous outcomes via meta-analysis, an odds ratio (OR) with 95 % confidence interval (CI) was calculated. A random-effects model was used describing the overall outcome in a forest plot. The I² statistic was used to assess heterogeneity with a result of I² > 50 % classified as significant. Overall, a result was considered significant with p < 0,05. Results: The initial electronic and manual research resulted in a total of 271 studies. Of these, 13 studies met the inclusion criteria for the systematic review, whereof five studies used MSCs as an orthobiologic treatment option. In three of the 13 studies administered autologous conditioned serum (ACS) and the remaining five studies used cellular autologous blood products with an increased platelet concentrate. Overall, two studies considered to be at a low risk of bias. After intra-articular treatment with orthobiologic therapeutics, an average improvement of 65 % in lameness was achieved, regardless the type of orthobiologic agent used. Eleven of the 13 studies showed a general positive effect after treatment with horses returning to their original performance level. Four of the 13 studies proved suitable for metaanalysis and were expressed using forest plot. The reduction in the degree of lameness between the treatment and control groups was compared. There was moderate heterogeneity in the long-term studies with an observation period of at least six months, I² = 55 %. All studies with an OR value > 1 favor the experimental group, which was appropriate here (OR 17,02; 95 % CI: 8,5474 to 33,8849 p < 0,0001). No study crossed the line into ineffectiveness, indicating that the treatment effect was considered similar among the studies. In the long-term follow-up, the use of intraarticularly administered orthobiologics led to a reduction in lameness in 73 % reduction in lameness, while lameness was reduced in 17 % in the control group. All documented cases (n = 15, 3 %) of the total 518 horses evaluated experienced transient adverse effects, also referred to as “joint flare”, an aseptic, inflammatory, and in most cases self-limiting joint reaction. One case resulted in exclusion from the study because blinding could not be maintained. Conclusion: According to the included studies, horses with naturally occurring OA after intraarticular treatment with orthobiologic therapeutics demonstrated a significantly lower degree of lameness at long-term follow-up compared to the control group, with a high level of safety of use. None of the horses showed significant side effects after treatment.:Inhaltsverzeichnis Abbildungsverzeichnis Abkürzungsverzeichnis Tabellenverzeichnis 1. Einleitung 2. Literaturübersicht 2.1 Die Osteoarthritis beim Pferd 2.1.1 Ätiologie beim Pferd 2.1.2 Pathomechanismus der Osteoarthritis 2.1.3 Osteoarthritis auf zellulärer Ebene 2.1.4 Histologische Studien zur Osteoarthritis beim Pferd 2.2 Regenerative/-Orthobiologische Therapien bei Osteoarthritis 2.2.1 Das therapeutische Konzept orthobiologischer Therapieansätze 2.2.2 Behandlungsoptionen der Osteoarthritis beim Pferd 2.2.3 Osteoarthritis: Das Pferd als Modelltier 2.3 Evidenzbasierte Medizin 2.3.1 Definition der evidenzbasierten Medizin 2.3.2 Das Systematische Review 2.3.3 Risk of bias 2.3.4 Die Meta-Analyse 3. Publikation 4. Diskussion 4.1 Methodische Diskussion der Studienauswertung 4.2 Kritische Begutachtung verschiedener OA-Modelle 4.3 Heterogenität der Studien 4.3.1 Diversität der Behandlungsmethoden 4.3.2 Uneinheitliche Definition des Erfolgsmerkmals und der Untersuchung 4.4 Kritik an randomisierten, kontrollierten Studien 4.5 Kritik an der Meta-Analyse 4.6 Schlussfolgerung 5. Zusammenfassung 6. Summary Literaturverzeichnis Danksagung

Axolotl uniquely generates blastema cells as a pool of progenitor/stem cells to restore an entire limb, a particular property that other organisms, such as humans, do not have. What underlies these differences? Is the main difference that cells residing at the amputation plane (in the stump) undergo reprogramming processes to re-enter the embryonic program, which allows developmental patterning to start, or are there fundamental differences? There is also a significant debate about whether regeneration occurs via stem cell differentiation or by dedifferentiation of mature limb tissue. The aim of my thesis was to address following questions: Are the cells in the blastema reprogrammed or differentiated to regenerate? Are the blastema cells genetically reactivated de novo during regeneration? How does the amputated limb exactly know which part of the limb needs to be regenerate? Using a novel technique of long-term genetic fate mapping, my team demonstrated that dedifferentiation in regenerated axolotl muscle tissue does not occur. Instead, PAX7+ satellite cells indeed play an important role during muscle regeneration in the axolotl limb. Surprisingly, this is in contrast to the newt, which regenerates muscle cells through a dedifferentiation process. Therefore, there is a fundamental difference that underlies the regenerative mechanism ((Sandoval-Guzman et al., 2014) [KR1]). This demonstrates that there is an unexpected diversity and flexibility of cellular mechanims used during limb regeneration, even among two closely related species. Finally, if one salamander species uses a mammalian regenerative strategy (Cornelison and Wold, 1997; Collins et al., 2005) involving stem cells and another uses a dedifferentiative strategy, this raises the question of whether there are other fundamental aspects of regeneration that could also be anomalous. This hypothesis is promising since there could be more than one possible mechanism to induce mammalian regeneration. The process of limb regeneration in principle seems to be more similar to those of limb development as historically assumed. We showed molecularly that embryonic players are reused during regeneration by reactivating the position- and tissue-specific developmental gene programs by using the newly isolated Twist sequences as early blastema cell markers ((Kragl et al., 2013) [KR2]). To gain insights into the molecular mechanisms of the P/D limb patterning in general, it was crucial to study the early patterning events of the resident progenitor/stem cells by using the specific blastema cell marker HoxA as a positional marker along the proximo-distal axis. Our HOXA protein analysis using high molecular and cellular resolution as well as transplantation assays demonstrated for the first time that axolotl limb blastema cells acquire their positional identity in a proximal to distal sequence. We found a hierarchy of cellular restrictions in positional identities. Amputation at the level of the upper arm showed that the blastema harbors cells, which convert to lower arm and hand. We observed ((Roensch et al., 2013) [KR3]) for the first time that intercalation- the intermediate element (lower arm) arises later from an interaction between the proximal and distal cells identities- does not occur. Intercalation, which has been an accepted model for a long time, is not the patterning mechanism underlying normal (without any manipulation) limb regeneration that is unique to axolotl. We further demonstrated, using the Hox genes as markers that positional identity is cell-type specific since their effects were confirmed to be present in the lateral plate mesoderm- derived cells of the limb. As our knowledge about limb blastemas expands concerning cell composition and molecular events controlling patterning, the similarity to development is becoming more and more clear. My work has resolved many ambiguities surrounding the molecularly identification of different types of blastema cells and how P/D limb patterning occurs during regeneration in comparison to development. It has highlighted the importance of combining high-resolution methods, such as in situ hybridizations, single-cell PCR (sc-PCR) of individual dissociated blastema cells and genetic labeling methods with grafting experiments to map cell fates in vivo. In addition to understanding the processes of regeneration, another long-term goal in the regenerative medicine field is to identify key molecules that trigger the regeneration of tissues. Recently, my colleague Takuji Sugiura (Sugiura et al., 2016) observed that an early event of blastema formation is the secretion of molecules like MLP (MARCKS-like protein), which induces wound-associated cell cycle re-entry. Such findings further increase the enthusiasm of biologists to understand the underlying principles of regeneration. By building our knowledge of the molecules and pathways that are involved in tissue regeneration, we increase the possibility of identifying a way to ‘activate’ regenerative processes in humans and thus reach the final goal of regenerative medicine, which is to use the concepts of cellular reprogramming, stem cell biology and tissue engineering to repair complex body structures.

Axolotl uniquely generates blastema cells as a pool of progenitor/stem cells to restore an entire limb, a particular property that other organisms, such as humans, do not have. What underlies these differences? Is the main difference that cells residing at the amputation plane (in the stump) undergo reprogramming processes to re-enter the embryonic program, which allows developmental patterning to start, or are there fundamental differences? There is also a significant debate about whether regeneration occurs via stem cell differentiation or by dedifferentiation of mature limb tissue. The aim of my thesis was to address following questions: Are the cells in the blastema reprogrammed or differentiated to regenerate? Are the blastema cells genetically reactivated de novo during regeneration? How does the amputated limb exactly know which part of the limb needs to be regenerate? Using a novel technique of long-term genetic fate mapping, my team demonstrated that dedifferentiation in regenerated axolotl muscle tissue does not occur. Instead, PAX7+ satellite cells indeed play an important role during muscle regeneration in the axolotl limb. Surprisingly, this is in contrast to the newt, which regenerates muscle cells through a dedifferentiation process. Therefore, there is a fundamental difference that underlies the regenerative mechanism ((Sandoval-Guzman et al., 2014) [KR1]). This demonstrates that there is an unexpected diversity and flexibility of cellular mechanims used during limb regeneration, even among two closely related species. Finally, if one salamander species uses a mammalian regenerative strategy (Cornelison and Wold, 1997; Collins et al., 2005) involving stem cells and another uses a dedifferentiative strategy, this raises the question of whether there are other fundamental aspects of regeneration that could also be anomalous. This hypothesis is promising since there could be more than one possible mechanism to induce mammalian regeneration. The process of limb regeneration in principle seems to be more similar to those of limb development as historically assumed. We showed molecularly that embryonic players are reused during regeneration by reactivating the position- and tissue-specific developmental gene programs by using the newly isolated Twist sequences as early blastema cell markers ((Kragl et al., 2013) [KR2]). To gain insights into the molecular mechanisms of the P/D limb patterning in general, it was crucial to study the early patterning events of the resident progenitor/stem cells by using the specific blastema cell marker HoxA as a positional marker along the proximo-distal axis. Our HOXA protein analysis using high molecular and cellular resolution as well as transplantation assays demonstrated for the first time that axolotl limb blastema cells acquire their positional identity in a proximal to distal sequence. We found a hierarchy of cellular restrictions in positional identities. Amputation at the level of the upper arm showed that the blastema harbors cells, which convert to lower arm and hand. We observed ((Roensch et al., 2013) [KR3]) for the first time that intercalation- the intermediate element (lower arm) arises later from an interaction between the proximal and distal cells identities- does not occur. Intercalation, which has been an accepted model for a long time, is not the patterning mechanism underlying normal (without any manipulation) limb regeneration that is unique to axolotl. We further demonstrated, using the Hox genes as markers that positional identity is cell-type specific since their effects were confirmed to be present in the lateral plate mesoderm- derived cells of the limb. As our knowledge about limb blastemas expands concerning cell composition and molecular events controlling patterning, the similarity to development is becoming more and more clear. My work has resolved many ambiguities surrounding the molecularly identification of different types of blastema cells and how P/D limb patterning occurs during regeneration in comparison to development. It has highlighted the importance of combining high-resolution methods, such as in situ hybridizations, single-cell PCR (sc-PCR) of individual dissociated blastema cells and genetic labeling methods with grafting experiments to map cell fates in vivo. In addition to understanding the processes of regeneration, another long-term goal in the regenerative medicine field is to identify key molecules that trigger the regeneration of tissues. Recently, my colleague Takuji Sugiura (Sugiura et al., 2016) observed that an early event of blastema formation is the secretion of molecules like MLP (MARCKS-like protein), which induces wound-associated cell cycle re-entry. Such findings further increase the enthusiasm of biologists to understand the underlying principles of regeneration. By building our knowledge of the molecules and pathways that are involved in tissue regeneration, we increase the possibility of identifying a way to ‘activate’ regenerative processes in humans and thus reach the final goal of regenerative medicine, which is to use the concepts of cellular reprogramming, stem cell biology and tissue engineering to repair complex body structures.

Cell transplantation and stem cell therapy are promising approaches for regenerative medicine and are of interest to researchers and clinicians worldwide. However, currently, no imaging technique that allows three-dimensional in vivo inspection of therapeutically administered cells in host tissues is available. Therefore, we investigate magnetomotive optical coherence tomography (MM-OCT) of cells labeled with magnetic particles as a potential noninvasive cell tracking method. We develop magnetomotive imaging of mesenchymal stem cells for future cell therapy monitoring. Cells were labeled with fluorescent iron oxide nanoparticles, embedded in tissue-mimicking agar scaffolds, and imaged using a microscope setup with an integrated MM-OCT probe. Magnetic particle-induced motion in response to a pulsed magnetic field of 0.2 T was successfully detected by OCT speckle variance analysis, and cross-sectional and volumetric OCT scans with highlighted labeled cells were obtained. In parallel, fluorescence microscopy and laser speckle reflectometry were applied as two-dimensional reference modalities to image particle distribution and magnetically induced motion inside the sample, respectively. All three optical imaging modalities were in good agreement with each other. Thus, magnetomotive imaging using iron oxide nanoparticles as cellular contrast agents is a potential technique for enhanced visualization of selected cells in OCT.

Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven, and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency, and treatment with zoledronic acid resulted in osteoclast function inhibition and failed tissue integration. Moreover, we identified the wound epithelium as a regulator of skeletal resorption, likely releasing signals involved in recruitment/differentiation of osteoclasts. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration.