Assembled Cell-Decorated Collagen (AC-DC) Fiber Bioprinted Implants with Musculoskeletal Tissue Properties Promote Functional Recovery in Volumetric Muscle Loss

Abstract

Musculoskeletal tissue injuries, including volumetric muscle loss (VML), are commonplace and often lead to permanent disability and deformation. Addressing this healthcare need, an advanced biomanufacturing platform, assembled cell-decorated collagen (AC-DC) bioprinting, is invented to rapidly and reproducibly create living biomaterial implants, using clinically relevant cells and strong, microfluidic wet-extruded collagen microfibers. Quantitative analysis shows that the directionality and distribution of cells throughout AC-DC implants mimic native musculoskeletal tissue. AC-DC bioprinted implants further approximate or exceed the strength and stiffness of human musculoskeletal tissue and exceed collagen hydrogel tensile properties by orders of magnitude. In vivo, AC-DC implants are assessed in a critically sized muscle injury in the hindlimb, with limb torque generation potential measured over 12 weeks. Both acellular and cellular implants promote functional recovery compared to the unrepaired group, with AC-DC implants containing therapeutic muscle progenitor cells promoting the highest degree of recovery. Histological analysis and automated image processing of explanted muscle cross-sections reveal increased total muscle fiber count, median muscle fiber size, and increased cellularization for injuries repaired with cellularized implants. These studies introduce an advanced bioprinting method for generating musculoskeletal tissue analogs with near-native biological and biomechanical properties with the potential to repair myriad challenging musculoskeletal injuries.

Keywords: AC-DC; bioprinting; collagen; microfibers; volumetric muscle loss.

Fig. 1.. Assembled Cell-decorated Collagen (AC-DC) bioprinting concept and design.

(A) Computer-aided design (CAD) model of our custom extrusion printhead and collection assembly with critical components identified. (B) Section view illustrating the cell seeding and implant biofabrication process. Collagen fiber passes through a seeding manifold, where it is uniformly coated with a cell suspension. The coated fiber is drawn onto a frame of arbitrary shape and dimensions and wrapped in parallel and on top of itself to form a 3D implant. (C) CAD model of a parallel fiber implant formed on a rigid frame with a multifunctional design. Frames maintain implant fidelity during culture, and implants are secured with sutures and easily removed from frames before further testing or implantation. (D) Photograph of printhead and collection assembly housed in a laminar flow hood during a single cell-solution bioprint.

Fig. 2.. Implant fidelity and cellularity.

(A) Three printed implants on a multifunctional frame before (top) and after (bottom) being secured with suture. (B) A larger printed implant on a custom frame geometry. (C) An implant is printed onto two lengths of sutures and held in tension by a custom frame. (D) Tweezers hold a printed implant after being secured with a suture and removed from a frame. (E) Fluorescence intensity indicating cell metabolic activity using the alamarBlue assay for implants printed with hMSCs after 1, 4, and 7 days of culture (n=6). (F) The transmitted light image of a printed implant showing striations indicative of densely packed parallel fiber after four days of culture. (G) Fluorescence image showing hMSCs distributed throughout and parallel to the fiber direction after four days of culture. Fluorescence images F-I show all cells with the cytoplasmic label DiD (green) and collagen fiber autofluorescence at 405 nm (blue), with images F and G additionally showing dead cell nuclei with EthD-1 (red). (H) Fluorescence image showing hMSCs attached to and distributed along three “building block” strands of collagen fiber after four days in culture. (I) Fluorescence images of a printed implant showing an initial distribution of cells after 1 day of culture and a confluent densely-cellularized implant after 26 days of culture. (J) Transmitted light image (left) and fluorescence image (right) of implants secured by suture showing dense cellular ingrowth of hMSCs between and on top of collagen fibers after 6 weeks of culture.

Fig. 3.. Quantitative cellular distribution.

(A) Typical field of view of a printed implant with rat muscle progenitor cells (MPCs) distributed throughout and aligned after 14 days in culture. The fluorescence image shows all cells with the cytoplasmic label DiD (green) and DAPI, with collagen fiber autofluorescence at 405 nm (blue). (B) Directionality analysis of the fiber-only component of (A), indicating highly parallel fiber. (C) Directionality analysis of the cell-only component of (A). (D) Image of (A) after processing for cell distribution analysis with cells shown in white, background in black, and transverse and longitudinal directions labeled. (E) Relative cellularity plotted along the transverse and (F) longitudinal directions, with nearly horizontal linear regression (dashed lines) and quantified uniformity measure U (ranging from 0 to 1), indicating a highly uniform distribution of cells.

Fig 4.. Implant mechanical properties.

AC-DC implants were printed with and without hMSCs and assessed after 1 day and 28 days in culture. (A) A custom 2-pin uniaxial tensile tensing setup was found to improve consistency in implant failure compared to traditional compression grips. (B) Typical stress-strain curves for each experimental group. (C) Measured cross-sectional areas suggest space between individual fiber centers from the HA coating and the fiber swelling as we previously reported (43). (D) Ultimate tensile stress (UTS). (E) Tangent modulus. (F) Strain at break. ^a Mean UTS and modulus of human ACL (59). ^b Mean UTS and modulus of the strongest portion of the human supraspinatus tendon (30). ^c Mean UTS and modulus of typical collagen gels used in tissue engineering (60). (All data n=10 per group per time point *P<0.05, **** P <0.0001 indicates significance).

Fig. 5.. Functional recovery in a rodent VML model.

(A) Creation of a VML injury measuring approximately 1 cm x 0.7 cm x 0.5 cm and weighing at a minimum 20% of the overall TA weight. (B) Acellular AC-DC implant inserted into the injury site and (C) sutured into the injury site with arrows indicating attachment points. (D) Fascia sutured overtop of the injury site to secure the implant in place further. (E) Animal weight pre-injury at 4, 8, and 12-weeks post-injury, corresponding to functional testing timepoints. (F) Weight of defects created for No repair, acellular implant, and cellular implant (NR, AI, and CI, respectively) experimental groups (p = 0.8, no significant difference). (G) Baseline torque generation pre-injury (p = 0.9, no significant difference). (H) Measured torque and (I) percent of baseline torque at 4, 8, and 12-weeks post-repair, indicating functional recovery facilitated by implant implantation. (All data n=7 per group per time point, *P<0.05, **P<0.01, ***P <0.001 indicates significance).

Fig. 6.. Histological assessment of the TA.

Representative H&E images of the TA muscle for (A) uninjured control, (B) no repair, (C) acellular implant, and (D) cellular implant experimental groups after 12 weeks. A black dashed line indicates the approximate area of defect creation. Green dashed ovals identify AC-DC implant locations. Magnified views of (E) acellular implant and (F) cellular implant locations with magnified windowed views showing cellular ingrowth and muscle fiber formation in the cellular implant location (yellow dashed oval). All scale bars are 1 mm unless otherwise noted.

Fig. 7.. Muscle fiber quantification using SMASH.

Representative laminin-stained sections of the TA muscle for (A) uninjured control, (B) no repair, (C) acellular implant, and (D) cellular implant experimental groups with dashed ovals indicating the approximate region of injury. (E-H) Colorized outputs from the software identifying individual muscle fibers within sections corresponding to (A-D), respectively. (I) Total fiber count, (J) median fiber cross-sectional area (FCSA), and (K) the product of fiber count and FCSA for uninured control (Ctrl), no repair (NR), acellular implant (AI), and cellular implant (CI) experimental groups. All scale bars are 1 mm. (All data n=7 per group per time point, *p<0.05 indicates significance).