Bioengineered dental tissues grown in the rat jaw

Abstract

Our long-term objective is to develop methods to form, in the jaw, bioengineered replacement teeth that exhibit physical properties and functions similar to those of natural teeth. Our results show that cultured rat tooth bud cells, seeded onto biodegradable scaffolds, implanted into the jaws of adult rat hosts and grown for 12 weeks, formed small, organized, bioengineered tooth crowns, containing dentin, enamel, pulp, and periodontal ligament tissues, similar to identical cell-seeded scaffolds implanted and grown in the omentum. Radiographic, histological, and immunohistochemical analyses showed that bioengineered teeth consisted of organized dentin, enamel, and pulp tissues. This study advances practical applications for dental tissue engineering by demonstrating that bioengineered tooth tissues can be regenerated at the site of previously lost teeth, and supports the use of tissue engineering strategies in humans, to regenerate previously lost and/or missing teeth. The results presented in this report support the feasibility of bioengineered replacement tooth formation in the jaw.

Figure 1. Dental cell proliferation in culture

(A) Analysis of cell number at day 0, and after 10 days in culture, revealed that enzyme concentrations of 0.4 mg/mL collagenase and 0.2 mg/mL dispase resulted in the highest cell yields. (B) Growth curves for digested tooth bud cell preparations confirmed that tooth buds digested with 0.4 mg/mL type I collagenase and 0.2 mg/mL dispase I exhibited the highest cell number after 10 days in culture. (C) Digital images of cultured tooth bud cells, prepared with the 0.4 mg/mL collagenase and 0.2 mg/mL dispase enzyme concentrations, after 2, 4, and 10 days in culture. Five independently isolated cell preparations were examined for each type of analysis (Table). Wilcoxon’s test revealed highly significant values of P = 0.0003. Scale bar is 2 mm.

Figure 2. Radiographic and histological analysis of harvested and sectioned 12-week experimental and control implants

(A) Negative control implant site. (B) Localized radiopaque areas (arrows) indicate mineralized tissue formation at the cell-seeded implant site. (C,E) H&E-stained positive control implanted 4-dpn tooth buds exhibited well-formed tooth structures, with organized crown and root structures. (D,F) Goldner’s stained 4-dpn tooth bud implants revealed characteristic blue-stained dentin, and brown-stained immature enamel. (G,I) H&E-stained bioengineered dental tissues present in jaw implants. (H,J) Goldner’s stained sections revealed blue-stained dentin, brown-stained immature enamel, and gray-stained mature enamel tissues. Unseeded, negative control scaffolds did not form dental tissues (data not shown). All 16 of the experimental cell-seeded implants and all of the 14 implanted tooth bud controls produced radiopaque dental tissues. None of the negative control unseeded scaffolds produced radiopaque mineralized tissue. Abbreviations: am, ameloblasts; b, bone; d, dentin; e, enamel; ime, immature enamel; me, mature enamel; od, odontoblasts; p, pulp; pd, pre-dentin. Scale bars are 100 µm.

Figure 3. Immunohistochemical (IHC) analysis of mandibular implant bioengineered tooth tissues. Bioengineered dental tissues were positive for amelogenin (AM)

(A,B), dentin sialophosphoprotein (DSPP) (D,E), periostin (PER) (G,H), and vimentum (VM) (J,K). Corresponding immunostaining of natural tooth tissues is shown (C,F,I,L). All isotype control immunostaining was negative. Three experimental PGA and three experimental PLGA cell-seeded scaffold implants were analyzed by IHC. Two negative control ‘scaffold alone’ controls and 2 positive control tooth bud implants were also analyzed by IHC. Abbreviations: AM, amelogenin; DSPP, dentin sialophosphoprotein; PER, periostin; VM, vimentum. Scale bars = 200 µm.