Micro-topographies as biomechanical niche for tendon tissue engineering

Tendons are mechanically competent bridges of the musculoskeletal system, acting as a highway for mechanical loading transmission between muscles and bones. Along with the intriguing limited knowledge on tendon biology, the unsatisfactory outcomes of current clinical treatments to tendon injuries have been driving the search for an alternative tissue engineered strategy envisioning tendon regeneration. Due to the mechanosensitive nature of tendon cells, replicating tendon niche in vitro requires mechanical stimuli for adequate cell functioning.

Vermeulen and colleagues at the MERLN Institute from the University of Maastricht, The Netherlands, hypothesize that micro-topographical architectures may generate topography-induced biomechanical cues to control the behavior of tenocytes, a population of cells from tendon tissue. The team of Professor Jan de Boer at the MERLN Institute previously developed a high-throughput screening platform with 2176 uniquely designed micro-topographies, the TopoChip [Unadkat et al. Proc. Natl. Acad. Sci. U.S.A.(2011) doi: 10.1073/pnas.1109861108]. Topographies at the surface of biomaterials provide instructive features to control cellular performance. The TopoChip platform is used to explore micro-topographical architectures as a biomechanical niche to support tenogenic cellular activities [Vermeulen et al., Acta Biomaterialia(2019) doi: 10.1016/j.actbio.2018.10.041]. 

The researchers describe that tenocytes suffer a phenotypic drift, exhibiting altered morphology and expressing lower levels of a major tenogenic marker, scleraxis (SCX), once under confluent culture conditions. Micro-topographies induce elevated SCX expression levels in such dedifferentiated tenocytes. Heterogeneous cellular morphological responses demonstrate that cell and nuclear area are key morphological features in directing SCX expression levels. TopoUnits inducing low and high SCX expression are identified. Cells in high micro-topographical pattern areas present a strong spreading morphology and belong to the highest SCX hits. 

Micro-topographies are selected for large area surface fabrication. These topographical cues induce elevated expression of tenogenic markers (SCX, Mohawk, Tenomodulin, Collagen type I) immediately after 48 h of culture, yet accompanied by increased levels of osteogenic and chondrogenic markers after 7 days.

The up-regulation of a mechanosensitive gene (EGR-1) after 2 h of culture suggests a biomechanical role for the micro-topographies. EGR-1 is indeed known to be up-regulated shortly upon mechanical stimulation. Thus, the high-throughput screening reveals a dynamic morphological remodeling of tenocytes in response to micro-topographies that is activated at the initial cell-substrate contacts. Of relevance to tendon tissue engineering, micro-topographies can be applied to directly guide cells. Tenocyte dedifferentiation and loss of SCX expression upon long-term culture (confluence culture) is not recovered in micro-topographies. Interestingly, repeated cell passaging (every 3 days) on the topographies leads to increased SCX expression and maintenance of elongated morphology, a characteristic feature of spindle-shaped tenocytes in their native in vivo niche.

"We believe that cells can read Braille, we just do not know the Braille language"Steven Vermeulen and Prof. Jan de Boer

The researchers explain that micro-topographical architectures provide tenogenic supportive cues to maintain the phenotypic profile of tenocytes. Multiple passaging of tenocytes is highlighted as presenting advantages over confluent culture conditions. This innovative platform holds potential to be used as alternative to standard culture substrates for cell expansion protocols in tendon tissue engineering.