Atanas Todorov

Engineering of a functional bone organ through endochondral ossification

Embryonic development, lengthening, and repair of most bones proceed by endochondral ossification, namely through formation of a cartilage intermediate. It was previously demonstrated that adult human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) can execute an endochondral program and ectopically generate mature bone. Here we hypothesized that hMSCs pushed through endochondral ossification can engineer a scaled-up ossicle with features of a “bone organ,” including physiologically remodeled bone, mature vasculature, and a fully functional hematopoietic compartment. Engineered hypertrophic cartilage required IL-1β to be efficiently remodeled into bone and bone marrow upon subcutaneous implantation. This model allowed distinguishing, by analogy with bone development and repair, an outer, cortical-like perichondral bone, generated mainly by host cells and laid over a premineralized area, and an inner, trabecular-like, endochondral bone, generated mainly by the human cells and formed over the cartilaginous template. Hypertrophic cartilage remodeling was paralleled by ingrowth of blood vessels, displaying sinusoid-like structures and stabilized by pericytic cells. Marrow cavities of the ossicles contained phenotypically defined hematopoietic stem cells and progenitor cells at similar frequencies as native bones, and marrow from ossicles reconstituted multilineage long-term hematopoiesis in lethally irradiated mice. This study, by invoking a “developmental engineering” paradigm, reports the generation by appropriately instructed hMSC of an ectopic “bone organ” with a size, structure, and functionality comparable to native bones. The work thus provides a model useful for fundamental and translational studies of bone morphogenesis and regeneration, as well as for the controlled manipulation of hematopoietic stem cell niches in physiology and pathology.

Osteoinductivity of engineered cartilaginous templates devitalized by inducible apoptosis

Significance It has been previously reported that hypertrophic cartilage tissues engineered from human mesenchymal stromal cells can efficiently remodel in vivo into bone organs, recapitulating developmental steps of endochondral ossification. We have here demonstrated that the extracellular matrix (ECM) of such engineered cartilage, even in the absence of a living cell component, retains frankly osteoinductive properties. The use of an apoptosis-driven devitalization technique revealed the importance of preserving the ECM integrity and, in particular, the embedded factors to trigger the regenerative process. Although exemplified in a skeletal context, our work outlines the general paradigm of cell-based but cell-free off-the-shelf materials capable of activating endogenous cells toward the formation of specific tissues. The role of cell-free extracellular matrix (ECM) in triggering tissue and organ regeneration has gained increased recognition, yet current approaches are predominantly based on the use of ECM from fully developed native tissues at nonhomologous sites. We describe a strategy to generate customized ECM, designed to activate endogenous regenerative programs by recapitulating tissue-specific developmental processes. The paradigm was exemplified in the context of the skeletal system by testing the osteoinductive capacity of engineered and devitalized hypertrophic cartilage, which is the primordial template for the development of most bones. ECM was engineered by inducing chondrogenesis of human mesenchymal stromal cells and devitalized by the implementation of a death-inducible genetic device, leading to cell apoptosis on activation and matrix protein preservation. The resulting hypertrophic cartilage ECM, tested in a stringent ectopic implantation model, efficiently remodeled to form de novo bone tissue of host origin, including mature vasculature and a hematopoietic compartment. Importantly, cartilage ECM could not generate frank bone tissue if devitalized by standard “freeze & thaw” (F&T) cycles, associated with a significant loss of glycosaminoglycans, mineral content, and ECM-bound cytokines critically involved in inflammatory, vascularization, and remodeling processes. These results support the utility of engineered ECM-based devices as off-the-shelf regenerative niches capable of recruiting and instructing resident cells toward the formation of a specific tissue.

Tissue decellularization by activation of programmed cell death

Decellularized tissues, native or engineered, are receiving increasing interest in the field of regenerative medicine as scaffolds or implants for tissue and organ repair. The approach, which offers the opportunity to deliver off-the-shelf bioactive materials without immuno-matching requirements, is based on the rationale that extracellular matrix (ECM)-presented cues can be potently instructive towards regeneration. However, existing decellularization protocols typically result in damage to the source ECM and do not allow the controlled preservation of its structural, biochemical and/or biomechanical features. Here we propose the deliberate activation of programmed cell death as a method to selectively target the cellular component of a tissue and thereby to preserve the integrity of the decellularized ECM. In the case of engineered tissues, the approach could be complemented by the use of (i) an immortalized cell line, engineered to undergo apoptosis upon exposure to a chemical inducer, and (ii) a perfusion bioreactor system, supporting efficient removal of cellular material. The combination of these tools may lead to the streamlined development of more appropriate materials, based on engineered and decellularized ECM and including a customized set of signals specifically designed to activate endogenous regenerative processes.

Tendon healing: an overview of physiology, biology, and pathology of tendon healing and systematic review of state of the art in tendon bioengineering

PurposeTendon injuries vary from acute rupture to chronic tendinopathy. For an optimal treatment of either condition, a profound knowledge is essential. Therefore, this article shall give an overview of physiology, biology, and pathology of tendon healing and state of the art in tendon bioengineering.MethodsFor a preferably comprehensive survey, the current literature listed in PubMed and published in English peer-reviewed journals (March 2013) was systematically reviewed for tendon healing and tendon bioengineering including cytokine modulation, autologous sources of growth factors, biomaterials, gene therapy, and cell-based therapy. No differentiation was made between clinical and preclinical in vitro investigations.ResultsTendon healing happens in certain stadiums of inflammation, formation, and remodelling. An additional process of “collagen recycling” close to the healing site has been described recently. With increasing comprehension of physiology and pathology of tendon healing, several promising approaches in tendon bioengineering using growth factors, biomaterials, gene therapy, or cell-based therapy are described. However, only some of these are already used routinely in clinics.ConclusionStrong and resistant tendons are crucial for a healthy musculoskeletal system. The new approaches in tendon bioengineering are promising to aid physiological tendon healing and thus resulting in a stronger and more resistant tendon after injury. The growing knowledge in this field will need to be further taken into clinical studies so that especially those patients with prolonged courses, revision surgery, or chronic tendinopathy and high-demanding patients, i.e., professional athletes would benefit.Level of evidenceII.

Implantation of Stromal Vascular Fraction Progenitors at Bone Fracture Sites: From a Rat Model to a First‐in‐Man Study

Stromal Vascular Fraction (SVF) cells freshly isolated from adipose tissue include osteogenic‐ and vascular‐progenitors, yet their relevance in bone fracture healing is currently unknown. Here, we investigated whether human SVF cells directly contribute to the repair of experimental fractures in nude rats, and explored the feasibility/safety of their clinical use for augmentation of upper arm fractures in elderly individuals. Human SVF cells were loaded onto ceramic granules within fibrin gel and implanted in critical nude rat femoral fractures after locking‐plate osteosynthesis, with cell‐free grafts as control. After 8 weeks, only SVF‐treated fractures did not fail mechanically and displayed formation of ossicles at the repair site, with vascular and bone structures formed by human cells. The same materials combined with autologous SVF cells were then used to treat low‐energy proximal humeral fractures in 8 patients (64‐84 years old) along with standard open reduction and internal fixation. Graft manufacturing and implantation were compatible with intraoperative settings and led to no adverse reactions, thereby verifying feasibility/safety. Biopsies of the repair tissue after up to 12 months, upon plate revision or removal, demonstrated formation of bone ossicles, structurally disconnected and morphologically distinct from osteoconducted bone, suggesting the osteogenic nature of implanted SVF cells. We demonstrate that SVF cells, without expansion or exogenous priming, can spontaneously form bone tissue and vessel structures within a fracture‐microenvironment. The gained clinical insights into the biological functionality of the grafts, combined with their facile, intra‐operative manufacturing modality, warrant further tests of effectiveness in larger, controlled trials. Stem Cells 2016;34:2956–2966

Anti-Inflammatory/Tissue Repair Macrophages Enhance the Cartilage-Forming Capacity of Human Bone Marrow-Derived Mesenchymal Stromal Cells

Macrophages are key players in healing processes. However, little is known on their capacity to modulate the differentiation potential of mesenchymal stem/stromal cells (MSC). Here we investigated whether macrophages (Mf) with, respectively, pro‐inflammatory and tissue‐remodeling traits differentially modulate chondrogenesis of bone marrow derived‐MSC (BM‐MSC). We demonstrated that coculture in collagen scaffolds of BM‐MSC with Mf derived from monocytes polarized with M‐CSF (M‐Mf), but not with GM‐CSF (GM‐Mf) resulted in significantly higher glycosaminoglycan (GAG) content than what would be expected from an equal number of BM‐MSC alone (defined as chondro‐induction). Moreover, type II collagen was expressed at significantly higher levels in BM‐MSC/M‐Mf as compared to BM‐MSC/GM‐Mf constructs, while type X collagen expression was unaffected. In order to understand the possible cellular mechanism accounting for chondro‐induction, developing monoculture and coculture tissues were digested and the properties of the isolated BM‐MSC analysed. We observed that as compared to monocultures, in coculture with M‐Mf, BM‐MSC decreased less markedly in number and exhibited higher clonogenic and chondrogenic capacity. Despite their chondro‐inductive effect in vitro, M‐Mf did not modulate the cartilage tissue maturation in subcutaneous pockets of nude mice, as evidenced by similar accumulation of type X collagen and calcified tissue. Our results demonstrate that coculture of BM‐MSC with M‐Mf results in synergistic cartilage tissue formation in vitro. Such effect seems to result from the survival of BM‐MSC with high chondrogenic capacity. Studies in an orthotopic in vivo model are necessary to assess the clinical relevance of our findings in the context of cartilage repair. J. Cell. Physiol. 230: 1258–1269, 2015.

Generation of a Bone Organ by Human Adipose-Derived Stromal Cells Through Endochondral Ossification

Recapitulation of endochondral ossification (ECO) (i.e., generation of marrow‐containing ossicles through a cartilage intermediate) has relevance to develop human organotypic models for bone or hematopoietic cells and to engineer grafts for bone regeneration. Unlike bone marrow‐derived stromal cells (also known as bone marrow‐derived mesenchymal stromal/stem cells), adipose‐derived stromal cells (ASC) have so far failed to form a bone organ by ECO. The goal of the present study was to assess whether priming human ASC to a defined stage of chondrogenesis in vitro allows their autonomous ECO upon ectopic implantation. ASC were cultured either as micromass pellets or into collagen sponges in chondrogenic medium containing transforming growth factor‐β3 and bone morphogenetic protein‐6 for 4 weeks (early hypertrophic templates) or for two additional weeks in medium supplemented with β‐glycerophosphate, l‐thyroxin, and interleukin1‐β to induce hypertrophic maturation (late hypertrophic templates). Constructs were implanted in vivo and analyzed after 8 weeks. In vitro, ASC deposited cartilaginous matrix positive for glycosaminoglycans, type II collagen, and Indian hedgehog. Hypertrophic maturation induced upregulation of type X collagen, bone sialoprotein, and matrix metalloproteinase13 (MMP13). In vivo, both early and late hypertrophic templates underwent cartilage remodeling, as assessed by MMP13‐ and tartrate‐resistant acid phosphatase‐positive staining, and developed bone ossicles, including bone marrow elements, although to variable degrees of efficiency. In situ hybridization for human‐specific sequences and staining with a human specific anti‐CD146 antibody demonstrated the direct contribution of ASC to bone and stromal tissue formation. In conclusion, despite their debated skeletal progenitor nature, human ASC can generate bone organs through ECO when suitably primed in vitro.

Engraftment of Prevascularized, Tissue Engineered Constructs in a Novel Rabbit Segmental Bone Defect Model

The gold standard treatment of large segmental bone defects is autologous bone transfer, which suffers from low availability and additional morbidity. Tissue engineered bone able to engraft orthotopically and a suitable animal model for pre-clinical testing are direly needed. This study aimed to evaluate engraftment of tissue-engineered bone with different prevascularization strategies in a novel segmental defect model in the rabbit humerus. Decellularized bone matrix (Tutobone) seeded with bone marrow mesenchymal stromal cells was used directly orthotopically or combined with a vessel and inserted immediately (1-step) or only after six weeks of subcutaneous “incubation” (2-step). After 12 weeks, histological and radiological assessment was performed. Variable callus formation was observed. No bone formation or remodeling of the graft through TRAP positive osteoclasts could be detected. Instead, a variable amount of necrotic tissue formed. Although necrotic area correlated significantly with amount of vessels and the 2-step strategy had significantly more vessels than the 1-step strategy, no significant reduction of necrotic area was found. In conclusion, the animal model developed here represents a highly challenging situation, for which a suitable engineered bone graft with better prevascularization, better resorbability and higher osteogenicity has yet to be developed.

Designing questionnaires: healthcare survey to compare two different response scales

BackgroundA widely discussed design issue in patient satisfaction questionnaires is the optimal length and labelling of the answering scale. The aim of the present study was to compare intra-individually the answers on two response scales to five general questions evaluating patients’ perception of hospital care.MethodsBetween November 2011 and January 2012, all in-hospital patients at a Swiss University Hospital received a patient satisfaction questionnaire on an adjectival scale with three to four labelled categories (LS) and five redundant questions displayed on an 11-point end-anchored numeric scale (NS). The scales were compared concerning ceiling effect, internal consistency (Cronbach’s alpha), individual item answers (Spearman’s rank correlation), and concerning overall satisfaction by calculating an overall percentage score (sum of all answers related to the maximum possible sum).ResultsThe response rate was 41% (2957/7158), of which 2400 (81%) completely filled out all questions. Baseline characteristics of the responders and non-responders were similar. Floor and ceiling effect were high on both response scales, but more pronounced on the LS than on the NS. Cronbach’s alpha was higher on the NS than on the LS. There was a strong individual item correlation between both answering scales in questions regarding the intent to return, quality of treatment and the judgement whether the patient was treated with respect and dignity, but a lower correlation concerning satisfactory information transfer by physicians or nurses, where only three categories were available in the LS. The overall percentage score showed a comparable distribution, but with a wider spread of lower satisfaction in the NS.ConclusionsSince the longer scale did not substantially reduce the ceiling effect, the type of questions rather than the type of answering scale could be addressed with a focus on specific questions about concrete situations instead of general questions. Moreover, the low correlation in questions about information provision suggests that only three possible response choices are insufficient. Further investigations are needed to find a more sensitive scale discriminating high-end ratings. Otherwise, a longitudinal within-hospital or a cross-sectional between-hospital comparison of patient care is questionable.

Fat-Derived Stromal Vascular Fraction Cells Enhance the Bone-Forming Capacity of Devitalized Engineered Hypertrophic Cartilage Matrix

Engineered and devitalized hypertrophic cartilage (HC) has been proposed as bone substitute material, potentially combining the features of osteoinductivity, resistance to hypoxia, capacity to attract blood vessels, and customization potential for specific indications. However, in comparison with vital tissues, devitalized HC grafts have reduced efficiency of bone formation and longer remodeling times. We tested the hypothesis that freshly harvested stromal vascular fraction (SVF) cells from human adipose tissue—which include mesenchymal, endothelial, and osteoclastic progenitors—enhance devitalized HC remodeling into bone tissue. Human SVF cells isolated from abdominal lipoaspirates were characterized cytofluorimetrically. HC pellets, previously generated by human bone marrow‐derived stromal cells and devitalized by freeze/thaw, were embedded in fibrin gel with or without different amounts of SVF cells and implanted either ectopically in nude mice or in 4‐mm‐diameter calvarial defects in nude rats. In the ectopic model, SVF cells added to devitalized HC directly contributed to endothelial, osteoblastic, and osteoclastic populations. After 12 weeks, the extent of graft vascularization and amount of bone formation increased in a cell‐number‐dependent fashion (up to, respectively, 2.0‐fold and 2.9‐fold using 12 million cells per milliliter of gel). Mineralized tissue volume correlated with the number of implanted, SVF‐derived endothelial cells (CD31+ CD34+ CD146+). In the calvarial model, SVF activation of HC using 12 million cells per milliliter of gel induced efficient merging among implanted pellets and strongly enhanced (7.3‐fold) de novo bone tissue formation within the defects. Our findings outline a bone augmentation strategy based on off‐the‐shelf devitalized allogeneic HC, intraoperatively activated with autologous SVF cells.