Written by T.K.H. Teh , P. Shi, X. Ren, J.H.P. Hui , S.L. Toh , and J.C.H. Goh

1. Ligament-to-bone Interface Tissue Regeneration Using a Functionalized Biphasic Silk Fibroin Scaffold Written by T.K.H. Teh, P. Shi, X. Ren, J.H.P. Hui, S.L. Toh, and J.C.H. Goh Reviewed by Brandon Smith

2. Background Information • Current prevailing method for repairing a torn Anterior Cruciate Ligament (ACL) is tendon autografts • Difficulties associated with this method • Limitation of donor site morbidity • Lack of graft integration with bone tunnel • Current Regenerative Engineering Attempts • Mesenchymal stem cell (MSC) seeded scaffolds for ligament-only regeneration • Successful in regenerating the ACL and epiligament with the existence of vasculature • Difficulties associated with this method • Limited bone insertion points • Significant decrease in load bearing capacity

3. Objective • Using a functionalized biphasic silk fibroin scaffold to properly form ACL along with bone tunnel tissues • On either end of the scaffold, hydroxyapatite nanoparticles (nHA) and bone morphogenic protein 2 (BMP2) were added to stimulate the regrowth of bone tunnel and enthesis • Central third of the scaffold left with the MSCs for ligament regeneration • Using rabbit and pig models to test this method

4. Methods and Materials • Scaffold Fabrication • In the Rabbit Model for in vivo characterization and in vitro characterization, they used knitted scaffolds with 240 fibroins, 60 x 20 mm • In the Pig model for in vivo characterization, they used scaffolds with 480 fibroins, 100 x 20 mm • Fabrication of osteogenic and tenogenic zones • Created three silk fibroin solutions: • Pure silk fibroin solution (2.6% w/v) • Aqueous silk fibroin (2.6% w/v) with nHA (.78 mg/end) • Aqueous silk fibroin (2.6% w/v), nHA (.78 mg/end), and BMP2 (29 ug/end) • Casted and froze multiple silk fibroin solutions on the knitted scaffold based on experimental group • Control group: Aqueous silk fibroin with nHA, pure aqueous silk fibroin, silk fibroin with nHA • Experimental group: aqueous silk fibroin with both, pure aqueous silk fibroin, silk fibroin with both

5. Methods and Materials • In Vitro Characterization • Cut the scaffolds at the phase boundaries to form three scaffolds with just pure silk fibroin, silk fibroin and nHA, and silk fibroin with both nHA and BMP2 • Rabbit MSCs were derived from bone marrow, seeded on the scaffolds, and cultured over 28 days • In Vivo Characterization • Rabbit Groups • Seeded the two types of bone scaffold, experimental and control, with rabbit MSCs and then were implanted into 48 rabbits • The rabbits were sacrificed at 2, 4, and 6 months • Pig Groups • Removed the ACL of the right knees of the pigs, then added the two types of scaffolds, having been seeded with pig MSCs. • The pigs were sacrificed at the 6 month mark

6. Methods and Materials: Critique • Major parts of this section were very vague by not giving full explanations of the full procedure • Never explains the characterization techniques used to acquire the data reported in the results sections • ex: “Characterization assays performed for cellular viability, proliferation, gene expression, and collagen deposition levels” • Scaffold characteristics • Only deeper explanation reported is the number of replicates used during each test

7. Results: Scaffold Morphology • This image shows that the scaffold contains interconnected pores • Critique • Caption says nHA and BMP2 are “securely incorporated”, but the image does not label either of those

8. Results: Elution Profile • This graph shows a similar response from physiological bone • Begins with large amounts of BMP2 at the onset • followed by smaller and smaller amounts for maintenance purposes and continued bone regrowth

9. Results: Gene Expression, Proliferation, and Collage Deposition • Shows the gene expression for the in vitro study. • the scaffold containing both nHA and BMP2 experienced the most growth in all of the 5 categories tested • Also shows the formation of collagen • Critique: • Only briefly explains what three of the five categories are

10. Results: Gross Observation of Excised Pig Knee Joints • Image A shows the regrowth of the ACL in the pig model from the control group • Image B shows the regrowth of the ACL in the pig model from the experimental group • Both are 6 month mark • Critique: • No image of what the ACL looked like before they began the whole procedure

11. Results: Micro – Ct images of excised Pig Femur • Micro CT images of the control (A) and experimental (D) pig femur • The box shows the original bone tunnel location • The arrow shows new calcified tissue deposition • Critique: • They give percentages of regrowth (6%, 68% for control and experimental respectively), but no scale bars or actual numbers

12. Results: Histological Images of Graft-Bone Interface • Images C and D show the histological images of the graft-bone interaction at 6 months post implantation for the control group and experimental group, respectively. • The arrows shows new calcified tissue deposition • The S is the silk scaffold remnant

13. Results: Mechanical Properties of Regenerated Anterior Cruciate Ligaments • This table shows the mechanical properties for • The original ACL • The previous scaffold method • The control scaffold group • The experimental scaffold group • The experimental is most like the original ACL in terms of both stiffness and maximum load for the TE ligament and ligament-bone interface • Critique: • Give a good control group in the original ACL for determining the effectiveness of the scaffold • Still no explanation of the tests performed

14. Conclusion and Overall Critique • Overall, the paper was very interesting, and could be deemed a success in terms of regenerating the bone tunnel and consequentially, a strong ACL • However the format was difficult to follow at some points • Would speak of the characterization techniques they used to find all the data, but never explained what those techniques were • Following some of the material in this article required previous knowledge of the subject, or outside research as to what some of the figures and methods referred to