Photoactivated composite biomaterial for soft tissue restoration in rodents and in humans

Abstract

Soft tissue reconstruction often requires multiple surgical procedures that can result in scars and disfiguration. Facial soft tissue reconstruction represents a clinical challenge because even subtle deformities can severely affect an individual's social and psychological function. We therefore developed a biosynthetic soft tissue replacement composed of poly(ethylene glycol) (PEG) and hyaluronic acid (HA) that can be injected and photocrosslinked in situ with transdermal light exposure. Modulating the ratio of synthetic to biological polymer allowed us to tune implant elasticity and volume persistence. In a small-animal model, implanted photocrosslinked PEG-HA showed a dose-dependent relationship between increasing PEG concentration and enhanced implant volume persistence. In direct comparison with commercial HA injections, the PEG-HA implants maintained significantly greater average volumes and heights. Reversibility of the implant volume was achieved with hyaluronidase injection. Pilot clinical testing in human patients confirmed the feasibility of the transdermal photocrosslinking approach for implantation in abdomen soft tissue, although an inflammatory response was observed surrounding some of the materials.

Fig. 1

Schematic showing transdermal photocrosslinking of composite soft tissue biomaterial. (A) Photopolymerizable poly(ethylene glycol) diacrylate–hyaluronic acid (PEG-HA) formulation is injected into the dermis. (B) The uncrosslinked solution (unorganized lines) is massaged into the desired shape. (C) Light-induced transdermal crosslinking to form the PEG-HA composite implant (organized lines).

Fig. 2

LED design, skin transmission, and PEG-HA crosslinking. (A) A unique 520- to 530-nm light-emitting diode (LED) array was designed. Scale bar, 200 μm. (B) The LED emission wavelengths were closely matched with the absorbance of the eosin Y photoinitiation system. (C) The array was designed to maximize light penetration through human skin for photocrosslinking the PEG-HA material. (D) The LED array was able to penetrate at least 4 mm into Fitzpatrick types I (pale white) and III (olive) human skin. (E) LED exposure time at an intensity of 43 mW/cm² was tailored to maximally polymerize the PEG-HA system, as measured by a plateau in elastic modulus. Data are means ± SEM (n = 4). (F) Swelling of PEG100-HA20 after 48 hours that had been crosslinked through different thicknesses of Fitzpatrick types I and III human skin. Data are means ± SEM (n = 3). (G and H) LED exposure of injected composites in the rat dorsum was followed immediately by thermal imaging of implants (outlined). (I and J) Microscopic images of HA5 particles (stained with blue ink for contrast) before (I) and after (J) photocrosslinking with PEG100. Scale bars, 200 μm. (K) Unmodified linear HA (10 mg/ml, 980 kD) crosslinked with PEG100. Scale bar, 200 μm.

Fig. 3

In vivo persistence of photocrosslinked PEG-HA composite implants. (A) Volumetric persistence of PEG-HA5 implants over 270 days with PEG at 100, 40, and 20 mg in the photocrosslinked implants. (B to D) Volumetric persistence for composite PEG100-HA implants compared with HA controls for at least 210 days: (B) PEG100-HA5 (n = 4), (C) PEG100-HA20 (n = 5), and (D) PEG100-HA24 (n = 20). Asterisks denote the first time point of significance between the two groups, with all subsequent points also being significant. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired Student’s t test. Data are means ± SEM. (E to G) HA24 and PEG100-HA24 implant volume and shape persistence were visualized with MRI immediately after polymerization (day 0) and after 47 and 110 days in vivo in a rat. (H and I) Three-dimensional reconstructions of implants in the rat dorsum clearly show the flattening of HA24 (HA) compared with PEG100-HA24 (PH) photocrosslinked implants at 47 and 110 days after polymerization (axes frame a transverse cut through the rat). (J) HA24 and PEG100-HA24 height persistence 100 days in vivo, as measured from MRI images in (E) to (G). *P < 0.05, unpaired Student’s t test. Data are means ± SEM.

Fig. 4

Histological characterization of photopolymerizable PEG-HA implants in the rat. PEG-HA composites were implanted deep into the superficial dorsal muscle in the subcutaneous space and monitored for 18 months. Location of the implants is noted by asterisks. Inflammatory capsules are noted with arrows. (A and B) PEG100-HA20 and corresponding HA20 control at day 2. (C to H) PEG100-HA5 and corresponding HA5 control at 1 month (C and D), 5 months (E and F), and 15 months (G and H). (I and J) PEG20-HA5 and PEG40-HA5 are shown at 15 months. (K) PEG100-HA20 was followed until 18 months in vivo. Scale bars, 100 μm.

Fig. 5

Pilot clinical study of photocrosslinked biosynthetic implants. (A) MRI of PEG100-HA24 photocrosslinked composite implants and HA24 controls in human abdominal skin at days 0 and 84. (B) Persistence of PEG100-HA24 and PEG100-HA20 implant heights compared with the respective HA24 and HA20 control injections. *P < 0.05, unpaired Student’s t test. Data are means ± SEM (n = 12).

Fig. 6

Immune response to implants in humans. PEG100-HA implants were examined ex vivo after 12 weeks. Representative images are shown for the composite implants (n = 32) as well as HA controls (n = 28). H&E shows tissue morphology, including the presence of pseudocapsules and inflammatory cells. Inflammatory cells were further characterized by immunostaining for T lymphocytes (CD3), helper T cells (CD4), cytotoxic T cells (CD8), and B lymphocytes (CD20). In all images, an asterisk denotes the implant location. Scale bars, 100 μm.